Nlrp3 modulators

ABSTRACT

The present invention provides compounds of Formula (I), wherein all of the variables are as defined herein. These compounds are modulators of NLRP3, which may be used as medicaments for the treatment of proliferative disorders, such as cancer in a subject (e.g., a human).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 62/959,208, filed Jan. 10, 2020; the content of which isherein incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure features chemical entities (e.g., a compound or apharmaceutically acceptable salt, and/or hydrate, and/or cocrystal,and/or drug combination of the compound) that modulate (e.g., agonizesor partially agonizes) NLRP3 that are useful, e.g., for treating acondition, disease or disorder in which an increase in NLRP3 signalingmay correct a deficiency in innate immune activity that contributes tothe pathology and/or symptoms and/or progression and/or treatmentrefractory state of the condition, disease or disorder (e.g., cancerswith low T-cell infiltration) in a subject (e.g., a human).

This disclosure also features compositions as well as other methods ofusing and making the same.

BACKGROUND

Nucleotide-binding oligomerization domain-like receptors (“NLRs”)include a family of intracellular receptors that detectpathogen-associated molecular patterns (“PAMPs”) and endogenousmolecules (see, e.g., Ting, J. P. Y. et al., “The NLR gene family: astandard nomenclature,” Immunity, 28(3):285-287, (2008)).

NLRPs represent a subfamily of NLRs that include a Pyrin domain and areconstituted by proteins such as NLRP1, NLRP3, NLRP4, NLRP6, NLRP7, andNLRP12. NLRPs are believed to be involved with the formation ofmultiprotein complexes termed inflammasomes (see, e.g., Chaput, C. etal., “NOD-like receptors in lung diseases,” Frontiers in Immunology, 4:article 393, (2013)). These complexes typically include one or two NLRproteins, the adapter molecule apoptosis associated speck-likecontaining a CARD domain (ASC) and pro-caspase-1 F (see, e.g.,Bauernfeind, F and Hornung, V. “Of inflammasomes and pathogens—sensingof microbes by the inflammasome,” EMBO Molecular Medicine, 5(6):814-826,(2013)).

One such inflammasome is formed by the NLRP3 scaffold, the ASC adaptorand pro-caspase-1 (see, e.g., Hirota, J. A., et al., “The airwayepithelium nucleotide-binding domain and leucine-rich repeat protein 3inflammasome is activated by urban particulate matter,” Journal ofAllergy and Clinical Immunology, 129(4):1116.e6-1125.e6, (2012)), andits expression is believed to be induced by inflammatory cytokines andTLR agonists in myeloid cells and human bronchial epithelial cells(Id.). The NLRP3 inflammasome is believed to mediate thecaspase-1-dependent conversion of pro-IL-1β and pro-IL-18 to IL-1β andIL-18. Further, IL-1β and IL-18 have potential in the treatment ofvarious types of cancer (see, e.g., Chen, L-C. et al., EMBO Mol Med.,4(12):1276-1293 (2012) and Tse, B. W-C. et al., PLoS One, 6(9):e24241(2011)). IL-18 has been shown to override resistance to checkpointinhibitors in colon cancer animal tumor models (see e.g., Ma, Z. et al.,Clin. Cancer Res. Jan. 11. (2016) DOI: 10.1158/1078-0432.CCR-15-1655).

SUMMARY

The invention is directed to compounds of Formula (I):

wherein all of the variables are as defined herein below.

Also within the scope of the invention are pharmaceutically acceptablesalts, stereoisomers, tautomers, and solvates of the compounds ofFormula (I).

The invention is also directed to pharmaceutical compositions comprisingone or more compounds of the invention. The invention is also directedto methods of treating cancer using one or more compounds of theinvention.

The invention also provides processes and intermediates for making thecompounds of Formula (I) or pharmaceutically acceptable salts,stereoisomers, tautomers, and solvates thereof.

The compounds of the invention may be used in therapy.

The compounds of the invention may be used for the manufacture of amedicament for the treatment of cancer.

The compounds of the invention can be used alone, in combination withother compounds of the present invention, or in combination with one ormore other agent(s).

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DETAILED DESCRIPTION Compounds of Invention

In a first aspect, the present invention provides, inter alia, acompound of Formula (I):

or a stereoisomer, a tautomer or a pharmaceutically acceptable saltthereof, wherein:

-   -   R¹ is independently selected from: H or C₁₋₆ alkyl substituted        with 0 to 3 halogen;    -   R², R³ and R⁵ are, at each occurrence, independently selected        from: H, halogen, cyano, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄        alkoxy, and C₁₋₄ haloalkoxy;    -   R⁴ is independently 5-membered heteroaryl including from 1 to 4        ring atoms are each independently selected from N, N(R^(a)), O,        and S, wherein the heteroaryl is substituted with from 0 to 3        R^(b);    -   R⁶ is independently selected from:

-   -   R⁷ is independently selected from: H, C₁₋₄ alkyl and —C(═O)R⁸;    -   R⁸ is independently selected from: C₂₋₄ alkynyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, C₁₋₄ alkyl substituted with 0 to 2 R^(c),        —(CH₂)₀₋₂—C₃₋₆ cycloalkyl substituted with 0 to 3 R^(d),        —(CH₂)₀₋₂-phenyl substituted with 0 to 3 R^(d), and        —(CH₂)₀₋₂-(5- to 6-membered heteroaryl including from 1 to 4        ring atoms are each independently selected from N, N(R^(a)), O,        and S, wherein the heteroaryl is substituted with from 0 to 3        R^(d));    -   R^(a) is, at each occurrence, independently selected from: H and        C₁₋₄ alkyl; and    -   R^(b), R^(c) and R^(d) are, at each occurrence, independently        selected from: halogen, OH, cyano, C₁₋₄ alkyl, C₁₋₄ haloalkyl,        C₁₋₄ alkoxy, and C₁₋₄ haloalkoxy.

In a second aspect, the present invention provides a compound of Formula(I), or a tautomer or a pharmaceutically acceptable salt thereof, withinthe scope of the first aspect, wherein:

-   -   R¹ is independently selected from: H or C₁₋₆ alkyl;    -   R², R³ and R⁵ are, at each occurrence, independently selected        from: H, halogen and C₁₋₄ alkyl;    -   R⁴ is independently pyrazolyl, thienyl or isothiazolyl;    -   R⁶ is independently selected from:

and

-   -   R⁷ is independently H or —C(═O)R⁸.

In a third aspect, the invention provides a compound of Formula (II):

or a stereoisomer, a tautomer or a pharmaceutically acceptable saltthereof, wherein:

-   -   R¹ is independently H or C₁₋₄ alkyl;    -   R⁶ is independently selected from:

-   -   R⁸ is independently selected from: C₂₋₄ alkynyl, C₁₋₄ alkoxy,        C₁₋₄ haloalkyl, phenyl, pyridyl, benzyl, C₁₋₄ alkyl substituted        with 0 to 1 R^(c), and C₃₋₆ cycloalkyl substituted with 0 to 2        R^(d);    -   R^(c) is independently C₁₋₄ alkoxy or cyano; and    -   R^(d) is independently selected from: F, cyano and C₁₋₄        haloalkyl.

In a fourth aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third aspect, wherein:

-   -   R⁶ is independently

In a fifth aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third aspect, wherein:

-   -   R⁶ is independently selected from:

In another aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third or fourth aspect, wherein:

-   -   R⁶ is independently

In another aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third or fourth aspect, wherein:

-   -   R⁶ is independently

In another aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third or fifth aspect, wherein:

-   -   R⁶ is independently

In another aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third or fifth aspect, wherein:

-   -   R⁶ is independently R⁸

In another aspect, the present invention provides a compound of Formula(II), or a stereoisomer, a tautomer or a pharmaceutically acceptablesalt thereof, within the scope of the third or fifth aspect, wherein:

-   -   R⁶ is independently

In one embodiment, R¹ is H. In another embodiment, R¹ is C₁₋₄ alkyl. Inanother embodiment, R¹ is ethyl or isopropyl.

In a sixth aspect, the invention provides a compound selected from theexemplified Examples 1 to 96 or a stereoisomer, a tautomer, or apharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a compound selectedfrom any subset list of compounds or a single compound from theexemplified examples within the scope of any of the above aspects.

The skilled artisan will recognize that some chemical structuresdescribed herein may be represented on paper by one or more otherresonance forms; or may exist in one or more other tautomeric forms,even when kinetically, the artisan recognizes that such tautomeric formsrepresent only a very small portion of a sample of such compound(s).

Such compounds are clearly contemplated within the scope of thisdisclosure, though such resonance forms or tautomers are not explicitlyrepresented herein.

Other Aspects and Embodiments of the Invention

In one aspect, methods for modulating (e.g., agonizing, partiallyagonizing, antagonizing) NLRP3 activity are featured that includecontacting NLRP3 with a chemical entity described herein (e.g., acompound described generically or specifically herein or apharmaceutically acceptable salt thereof or compositions containing thesame).

In preferred embodiments, methods for modulating NLRP3 activity areagonizing and partially agonizing. In certain embodiments, methods formodulating NLRP3 activity are agonizing. In certain embodiments, methodsfor modulating NLRP3 activity are partially agonizing. Methods includein vitro methods, e.g., contacting a sample that includes one or morecells comprising NLRP3 (e.g., THP-1 cells) with the chemical entity.Methods can also include in vivo methods; e.g., administering thechemical entity to a subject (e.g., a human) having a disease in whichan increase in NLRP3 signaling may correct a deficiency in innate immuneactivity that contributes to the pathology and/or symptoms and/orprogression of the disease (e.g., cancer; e.g., a refractory cancer).

In some embodiments, compounds of the invention are useful for treatinga condition, disease or disorder in which a decrease in NLRP3 activity(e.g., a condition, disease or disorder associated with repressed orimpaired NLRP3 signaling) contributes to the pathology and/or symptomsand/or progression of the condition, disease or disorder (e.g., cancer)in a subject (e.g., a human).

A cancer is said to be refractory when it does not respond to (or isresistant to) cancer treatment. Refractory cancer is also known asresistant cancer.

In another aspect, methods of treating cancer are featured that includeadministering to a subject in need of such treatment an effective amountof a chemical entity described herein (e.g., a compound describedgenerically or specifically herein or a pharmaceutically acceptable saltthereof or compositions containing the same). In some embodiments, thecancer may be a refractory cancer.

In a further aspect, methods of treatment of a disease in which anincrease in NLRP3 signaling may correct a deficiency in innate immuneactivity that contributes to the pathology and/or symptoms and/orprogression of the disease are featured that include administering to asubject in need of such treatment an effective amount of a chemicalentity described herein (e.g., a compound described generically orspecifically herein or a pharmaceutically acceptable salt thereof orcompositions containing the same).

In another aspect, methods of treatment are featured that includeadministering to a subject having a disease in which an increase inNLRP3 signaling may correct a deficiency in innate immune activity thatcontributes to the pathology and/or symptoms and/or progression of thedisease an effective amount of a chemical entity described herein (e.g.,a compound described generically or specifically herein or apharmaceutically acceptable salt thereof or compositions containing thesame).

In a further aspect, methods of treatment are featured that includeadministering to a subject a chemical entity described herein (e.g., acompound described generically or specifically herein or apharmaceutically acceptable salt thereof or compositions containing thesame), wherein the chemical entity is administered in an amounteffective to treat a disease in which an increase in NLRP3 signaling maycorrect a deficiency in innate immune activity that contributes to thepathology and/or symptoms and/or progression of the disease, therebytreating the disease.

Embodiments can include one or more of the following features.

The chemical entity can be administered in combination with one or moreadditional cancer therapies (e.g., surgery, radiotherapy, chemotherapy,toxin therapy, immunotherapy, cryotherapy or gene therapy, or acombination thereof, e.g., cancer therapies that include administeringone or more (e.g., two, three, four, five, six, or more) additionalanti-cancer agents. Non-limiting examples of additional anti-canceragents (chemotherapeutic agents) are selected from an alkylating agent(e.g., cisplatin, carboplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide and/or oxaliplatin); an anti-metabolite (e.g.,azathioprine and/or mercaptopurine); a terpenoid (e.g., a vinca alkaloidand/or a taxane; e.g., Vincristine, Vinblastine, Vinorelbine and/orVindesine, Taxol, Paclitaxel and/or Docetaxel); a topoisomerase (e.g., atype I topoisomerase and/or a type 2 topoisomerase; e.g., camptothecins,such as irinotecan and/or topotecan; amsacrine, etoposide, etoposidephosphate and/or teniposide); a cytotoxic antibiotic (e.g., actinomycin,anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin,epirubicin, bleomycin, plicamycin and/or mitomycin); a hormone (e.g., alutenizing hormone releasing hormone agonist; e.g., leuprolidine,goserelin, triptorelin, histrelin, bicalutamide, flutamide and/ornilutamide); an antibody (e.g., Abciximab, Adalimumab, Alemtuzumab,Atlizumab, Basiliximab, Belimumab, Bevacizumab, Brentuximab vedotin,Canakinumab, Cetuximab, Certolizumab pegol, Daclizumab, Denosumab,Eculizumab, Efalizumab, Gemtuzumab, Golimumab, Ibritumomab tiuxetan,Infliximab, Ipilimumab, Muromonab-CD3, Natalizumab, Ofatumumab,Omalizumab, Palivizumab, Panitummuab, Ranibizumab, Rituximab,Tocilizumab, Tositumomab and/or Trastuzumab); an anti-angiogenic agent;a cytokine; a thrombotic agent; a growth inhibitory agent; ananti-helminthic agent; and an immune checkpoint inhibitor that targetsan immune checkpoint receptor selected from CTLA-4, PD-1, PD-L1,PD-1—PD-L1, PD-1—PD-L2, T cell immunoglobulin and mucin 3 (TIM3 orHAVCR2), Galectin 9—TIM3, Phosphatidylserine—TIM3, lymphocyte activationgene 3 protein (LAG3), MHC class II—LAG3, 4-1BB-4-1BB ligand, OX40-OX40ligand, GITR, GITR ligand—GITR, CD27, CD70-CD27, TNFRSF25,TNFRSF25-TL1A, CD40L, CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM—BTLA,HVEM—CD160, HVEM—LIGHT, HVEM-BTLA-CD160, CD80, CD80—PDL-1, PDL2—CD80,CD244, CD48—CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA,TMIGD2, HHLA2-TMIGD2, Butyrophilins, including BTNL2, Siglec family,TIGIT and PVR family members, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICAand MICB, CD244, CD28, CD86—CD28, CD86—CTLA, CD80—CD28,Phosphatidylserine, TIM3, Phosphatidylserine—TIM3, SIRPA-CD47, VEGF,Neuropilin, CD160, CD30, and CD155 (e.g., CTLA-4 or PD1 or PD-L1) andother immunomodulatory agents, such as interleukin-2 (IL-2), indoleamine2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFβ), CD39,CD73 Adenosine-CD39-CD73, and CXCR4-CXCL12.

The subject can have cancer; e.g., the subject has undergone and/or isundergoing and/or will undergo one or more cancer therapies.

Non-limiting examples of cancer include acute myeloid leukemia,adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer,appendix cancer, teratoid/rhabdoid tumor, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancer, brain cancer, breast cancer,bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer,chordoma, chronic lymphocytic leukemia, chronic myeloproliferativeneoplasm, colon cancer, colorectal cancer, craniopharyngioma,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, eye cancer, fallopian tube cancer,gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor, germ cell tumor, hairy cell leukemia, head and neckcancer, heart cancer, liver cancer, hypopharngeal cancer, pancreaticcancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia,lip and oral cavity cancer, lung cancer, melanoma, Merkel cellcarcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma,ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer,rectal cancer, salivary gland cancer, skin cancer, small intestinecancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, and vulvarcancer.

In other embodiments, the mammal has been identified as having a canceror an infectious disease. Representative infectious diseases include,without limitation, Acinobacter infection, actinomycosis, Africansleeping sickness, acquired immunodeficiency syndrome, amebiasis,anaplasmosis, anthrax, Arcanobacterium haemolyticum infection, Argentinehemorrhagic fever, ascariasis, aspergillosis, astrovirus infection,babesiosis, Bacillus cereus infection, bacterial pneumonia, bacterialvaginosis, Bacteroides infection, balantidiasis, Baylisascarisinfection, BK virus infection, black piedra, Blastocystic hominisinfection, blastomycosis, Bolivian hemorrhagic fever, botulism,Brazilian hemorrhagic fever, brucellosis, bubonic plaque, Burkholderiinfection, Buruli ulcer, Calicivirus infection, camptobacteriosis,candidiasis, cat-scratch disease, cellulitis, Chagas disease, chancroid,chickenpox, chikungunya, chlamydia, Chlamydophila pneumoniae infection,cholera, chromoblastomycosis, clonorchiasis, Clostridium difficileinfection, coccidioidomycosis, Colorado tick fever, common cold,Creutzfeldt-Jakob disease, Crimean-Congo hemorrhagic fever,crytococcosis, cryptosporidiosis, cutaneous larva migrans,cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever,Desmodesmus infection, deintamoebiasis, diphtheria, diphyllobothriasis,dracunculiasis, ebola hemorrhagic fever, echinococcosis, ehrlichiosis,enterobiasis, Enterococcus infection, Enterovirus infection, epidemictyphus, erythema infection, exanthema subitum, fasciolopsiasis,fasciolosis, fatal familial insomnia, filariasis, food poisoning byClostridium myonecrosis, free-living amebic infection, Fusobacteriuminfection, gas gangrene, geotrichosis, Gerstmann-Straussler-Scheinkersyndrome, giardiasis, glanders, gnathostomiasis, gonorrhea, granulomainguinale, Group A streptococcal infection, Group B streptococcalinfection, Haemophilus influenzae infection, hand foot and mouthdisease, hantavirus pulmonary syndrome, Heartland virus disease,Heliobacter pylori infection, hemolytic-uremic syndrome, hemorrhagicfever with renal syndrome, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworminfection, human bocavirus infection, human ewingii ehrlichiosis, humangranulocyte anaplasmosis, human metapneuomovirus infection, humanmonocytic ehrlichiosis, human papillomavirus infection, humanparainfluenza virus infection, hymenolepiasis, Epstein-Barr virusinfectious mononucleosis, influenza, isosporiasis, Kawasaki disease,keratitis, Kingella kingae infection, kuru, lassa fever, Legionnaires'disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis,listeriosis, lyme disease, lymphatic filariasis, lymphocyticchoriomeningitis, malaria, Marburg hemorrhagic fever, measles, MiddleEast respiratory syndrome, melioidosis, meningitis, meningococcaldisease, metagonimiasis, microsporidiosis, molluscum contagiosum,monkeypox, mumps, murine typhus, mycoplasma pneumonia, mycetoma,myiasis, neonatal conjunctivitis, variant Creutzfeldt-Jakob disease,nocardiosis, onchocerciasis, paracoccidioidomycosis, paragonimiasis,pasteurellosis, pediculosis capitis, pediculosis corporis, pediculosispubis, pelvic inflammatory disease, pertussis, plague, pneumonia,poliomyelitis, Prevotella infection, primary amoebicmeningoencephalitis, progressive multifocal leukoencephalopathy,psittacosis, Q fever, rabies, relapsing fever, respiratory syncytialvirus infection, rhinosporidiosis, rhinovirus infection, rickettsialinfection, rickettsialpox, Rift Valley Fever, Rocky Mountain spottedfever, rotavirus infection, rubella, salmonellosis, severe acuterespiratory syndrome, scabies, schistosomiasis, sepsis, shigellosis,shingles, smallpox, sporothrichosis, staphylococcal food poisoning,staphylococcal infection, strongyloidiasis, subacute sclerosingpanencephalitis, syphilis, taeniasis, tetanus, tinea barabe, tineacapitis, tinea corporis, tinea cruris, tinea manum, tinea nigra, tineapedis, tinea unguium, tinea versicolor, toxocariasis, trachoma,toxoplasmosis, trichinosis, trichomoniasis, trichuriasis, tuberculosis,tularemia, typhoid fever, Ureaplasma urealyticum infection, valleyfever, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever,white piedra, Yersiniapsuedotuberculosis infection, yersiniosis, yellowfever, and zygomycosis.

The chemical entity can be administered intratumorally.

The chemical entity can be administered systemically (including but notlimited to orally, subcutaneously, intramuscular, intravenously).

The methods can further include identifying the subject.

Other embodiments include those described in the Detailed Descriptionand/or in the claims.

Definitions

To facilitate understanding of the disclosure set forth herein, a numberof additional terms are defined below. Generally, the nomenclature usedherein and the laboratory procedures in organic chemistry, medicinalchemistry, and pharmacology described herein are those well-known andcommonly employed in the art. Unless defined otherwise, all technicaland scientific terms used herein generally have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

Unless specifically stated otherwise herein, references made in thesingular may also include the plural. For example, “a” and “an” mayrefer to either one, or one or more.

Unless otherwise indicated, any heteroatom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

For purposes of clarity and in accordance with standard convention inthe art, the symbol

is used in formulas and tables to show the bond that is the point ofattachment of the moiety or substituent to the core/nucleus of thestructure.

Additionally, for purposes of clarity, where a substituent has a dash(-) that is not between two letters or symbols; this is used to indicatea point of attachment for a substituent. For example, —OCH₃ is attachedthrough the oxygen atom.

As used herein, the term “NLRP3” is meant to include, withoutlimitation, nucleic acids, polynucleotides, oligonucleotides, sense andantisense polynucleotide strands, complementary sequences, peptides,polypeptides, proteins, homologous and/or orthologous NLRP3 molecules,isoforms, precursors, mutants, variants, derivatives, splice variants,alleles, different species, and active fragments thereof.

An “agonist” of NLRP3 includes compounds that, at the protein level,directly bind or modify NLRP3 such that an activity of NLRP3 isincreased, e.g., by activation, stabilization, altered distribution, orotherwise.

Certain compounds described herein that agonize NLRP3 to a lesser extentthan a NLRP3 full agonist can function in assays as antagonists as wellas agonists. These compounds antagonize activation of NLRP3 by a NLRP3full agonist because they prevent the full effect of NLRP3 interaction.However, the compounds also, on their own, activate some NLRP3 activity,typically less than a corresponding amount of the NLRP3 full agonist.Such compounds may be referred to as “partial agonists of NLRP3”.

In some embodiments, the compounds described herein are agonists (e.g.full agonists) of NLRP3. In other embodiments, the compounds describedherein are partial agonists of NLRP3.

Generally, a receptor exists in an active (Ra) and an inactive (Ri)conformation. Certain compounds that affect the receptor can alter theratio of Ra to Ri (Ra/Ri). For example, a full agonist increases theratio of Ra/Ri and can cause a “maximal”, saturating effect. A partialagonist, when bound to the receptor, gives a response that is lower thanthat elicited by a full agonist (e.g., an endogenous agonist). Thus, theRa/Ri for a partial agonist is less than for a full agonist. However,the potency of a partial agonist may be greater or less than that of thefull agonist.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

“API” refers to an active pharmaceutical ingredient.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of a chemical entity (e.g., acompound exhibiting activity as a mitochondrial uncoupling agent or apharmaceutically acceptable salt and/or hydrate and/or cocrystalthereof; e.g., a compound, such as niclosamide or a pharmaceuticallyacceptable salt and/or hydrate and/or cocrystal thereof; e.g., acompound, such as a niclosamide analog, or a pharmaceutically acceptablesalt and/or hydrate and/or cocrystal thereof) being administered whichwill relieve to some extent one or more of the symptoms of the diseaseor condition being treated. The result includes reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga compound as disclosed herein required to provide a clinicallysignificant decrease in disease symptoms. An appropriate “effective”amount in any individual case is determined using any suitabletechnique, such as a dose escalation study.

The term “excipient” or “pharmaceutically acceptable excipient” means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, carrier, solvent, or encapsulatingmaterial. In one embodiment, each component is “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof a pharmaceutical formulation, and suitable for use in contact withthe tissue or organ of humans and animals without excessive toxicity,irritation, allergic response, immunogenicity, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio. See,e.g., Remington: The Science and Practice of Pharmacy, 22nd Edition,Pharmaceutical Press, London, UK (2012); Handbook of PharmaceuticalExcipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and theAmerican Pharmaceutical Association: (2009); Handbook of PharmaceuticalAdditives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: (2007);Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRCPress LLC: Boca Raton, FL, (2009).

The term “pharmaceutically acceptable salt” refers to a formulation of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In certain instances, pharmaceuticallyacceptable salts are obtained by reacting a compound described herein,with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. In some instances,pharmaceutically acceptable salts are obtained by reacting a compoundhaving acidic group described herein with a base to form a salt such asan ammonium salt, an alkali metal salt, such as a sodium or a potassiumsalt, an alkaline earth metal salt, such as a calcium or a magnesiumsalt, a salt of organic bases such as dicyclohexylamine,N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts withamino acids such as arginine, lysine, and the like, or by other methodspreviously determined. The pharmacologically acceptable salt is notspecifically limited as far as it can be used in medicaments. Examplesof a salt that the compounds described hereinform with a base includethe following: salts thereof with inorganic bases such as sodium,potassium, magnesium, calcium, and aluminum; salts thereof with organicbases such as methylamine, ethylamine and ethanolamine; salts thereofwith basic amino acids such as lysine and ornithine; and ammonium salt.The salts may be acid addition salts, which are specifically exemplifiedby acid addition salts with the following: mineral acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,nitric acid, and phosphoric acid:organic acids such as formic acid,acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid,fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid,citric acid, methanesulfonic acid, and ethanesulfonic acid; acidic aminoacids such as aspartic acid and glutamic acid.

The term “pharmaceutical composition” refers to a mixture of a compounddescribed herein with other chemical components (referred tocollectively herein as “excipients”), such as carriers, stabilizers,diluents, dispersing agents, suspending agents, and/or thickeningagents. The pharmaceutical composition facilitates administration of thecompound to an organism. Multiple techniques of administering a compoundexist in the art including, but not limited to: rectal, oral,intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topicaladministration.

The term “subject” refers to an animal, including, but not limited to, aprimate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat,rabbit, rat, or mouse. The terms “subject” and “patient” are usedinterchangeably herein in reference, for example, to a mammaliansubject, such as a human.

The terms “treat,” “treating,” and “treatment,” in the context oftreating a disease or disorder, are meant to include alleviating orabrogating a disorder, disease, or condition, or one or more of thesymptoms associated with the disorder, disease, or condition; or toslowing the progression, spread or worsening of a disease, disorder orcondition or of one or more symptoms thereof. The “treatment of cancer”,refers to one or more of the following effects: (1) inhibition, to someextent, of tumor growth, including, (i) slowing down and (ii) completegrowth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion and/or (8) relief, to some extent, of the severity or number ofone or more symptoms associated with the disorder.

The term “halo” or “halogen” refers to fluoro (F), chloro (Cl), bromo(Br), or iodo (I).

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁₋₁₀ indicates that the group may have from 1 to 10(inclusive) carbon atoms in it. Non-limiting examples include methyl,ethyl, iso-propyl, tert-butyl, n-hexyl.

The term “alkylene” refers to a branched or unbranched divalent alkyl(e.g., —CH₂—).

The term “haloalkyl” refers to an alkyl, in which one or more hydrogenatoms is/are replaced with an independently selected halo.

The term “alkoxy” refers to an —O-alkyl radical (e.g., —OCH₃).

The term “haloalkoxy” refers to an —O-haloalkyl group as defined abovewith the indicated number of carbon atoms attached through an oxygenbridge. For example, “C₁₋₆ haloalkoxy”, is intended to include C₁, C₂,C₃, C₄, C₅, and C₆ haloalkoxy groups. Examples of haloalkoxy include,but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, andpentafluorothoxy.

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “aromatic” refers generally to a ring that includes a cyclicarray of resonance-stabilized 4n+2 pi electrons, wherein n is an integer(e.g., 1 or 2). Aromatic moieties include aryl and heteroaryl groups.The term “nonaromatic” describes any moiety that does not fall withinthe definition of “aromatic”.

The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic, or14-carbon tricyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atomsof each ring may be substituted by a substituent, and wherein the ringcomprising a monocyclic radical is aromatic and wherein at least one ofthe fused rings comprising a bicyclic or tricyclic radical is aromatice.g. tetrahydronaphthyl. Examples of aryl groups also include phenyl,naphthyl and the like.

The term “cycloalkyl” as used herein includes saturated cyclichydrocarbon groups having 5 to 12 carbons, preferably bicyclic ortricyclic and 5 to 10 carbons, wherein the cycloalkyl group may be aspirocyclic ring or contain one or more bridged linker(s), and may beoptionally substituted. The term “cycloalkylene” as used herein refersto divalent cycloalkyl.

The term “heterocycloalkyl” as used herein includes saturated cyclichydrocarbon groups having 5 to 12 ring atoms, preferably bicyclic 5 to10 ring atoms, including from 1 to 4 ring atoms are each independentlyselected from N (or substituted N), O, and S, wherein theheterocycloalkyl may be a spirocyclic ring or contain a bridged linker,and may be optionally substituted. The term “heterocycloalkylene” asused herein refers to divalent heterocycloalkyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent, and whereinthe ring comprising a monocyclic radical is aromatic and wherein atleast one of the fused rings comprising a bicyclic or tricyclic radicalis aromatic (but does not have to be a ring which contains a heteroatom,e.g. tetrahydroisoquinolinyl. Examples of heteroaryl groups also includepyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl,thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

In addition, atoms making up the compounds of the present embodimentsare intended to include all isotopic forms of such atoms. Isotopes, asused herein, include those atoms having the same atomic number butdifferent mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include ¹³C and ¹⁴C.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the description anddrawings, and from the claims.

This disclosure features chemical entities (e.g., a compound or apharmaceutically acceptable salt, and/or hydrate, and/or cocrystal,and/or drug combination of the compound) that modulate (e.g., agonizesor partially agonizes) NLRP3 that are useful, e.g., for treating acondition, disease or disorder in which an increase in NLRP3 signalingmay correct a deficiency in innate immune activity (e.g., a condition,disease or disorder associated with an insufficient immune response)that contributes to the pathology and/or symptoms and/or progression ofthe condition, disease or disorder (e.g., cancer) in a subject (e.g., ahuman). This disclosure also features compositions as well as othermethods of using and making the same.

Pharmaceutical Compositions and Administration

In some embodiments, a chemical entity (e.g., a compound that modulates(e.g., agonizes or partially agonizes) NLRP3, or a pharmaceuticallyacceptable salt, and/or hydrate, and/or cocrystal, and/or drugcombination thereof) is administered as a pharmaceutical compositionthat includes the chemical entity and one or more pharmaceuticallyacceptable excipients, and optionally one or more additional therapeuticagents as described herein.

In some embodiments, a pharmaceutical composition comprising a compoundof the present invention or a salt thereof, and one or morepharmaceutically acceptable excipients. In certain embodiments, apharmaceutical composition comprising a compound of the presentinvention or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients. In certain embodiments, apharmaceutical composition comprising a therapeutically effective amountof a compound of the present invention or a pharmaceutically acceptablesalt thereof, and one or more pharmaceutically acceptable excipients.

In some embodiments, the chemical entities can be administered incombination with one or more conventional pharmaceutical excipients.Pharmaceutically acceptable excipients include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifyingdrug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol1000 succinate, surfactants used in pharmaceutical dosage forms such asTweens, poloxamers or other similar polymeric delivery matrices, serumproteins, such as human serum albumin, buffer substances such asphosphates, tris, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethyl cellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, andwool fat. Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemicallymodified derivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives canalso be used to enhance delivery of compounds described herein. Dosageforms or compositions containing a chemical entity as described hereinin the range of 0.005% to 100% with the balance made up from non-toxicexcipient may be prepared. The contemplated compositions may contain0.001%-100% of a chemical entity provided herein, in one embodiment0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington: TheScience and Practice of Pharmacy, 22^(nd) Edition (Pharmaceutical Press,London, U K. 2012).

Routes of Administration and Composition Components

In some embodiments, the chemical entities described herein or apharmaceutical composition thereof can be administered to subject inneed thereof by any accepted route of administration. Acceptable routesof administration include, but are not limited to, buccal, cutaneous,endocervical, endosinusial, endotracheal, enteral, epidural,interstitial, intra-abdominal, intra-arterial, intrabronchial,intrabursal, intracerebral, intracisternal, intracoronary, intradermal,intraductal, intraduodenal, intradural, intraepidermal, intraesophageal,intragastric, intragingival, intraileal, intralymphatic, intramedullary,intrameningeal, intramuscular, intraovarian, intraperitoneal,intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial,intratesticular, intrathecal, intratubular, intratumoral, intrauterine,intravascular, intravenous, nasal, nasogastric, oral, parenteral,percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous,sublingual, submucosal, topical, transdermal, transmucosal,transtracheal, ureteral, urethral and vaginal. In certain embodiments, apreferred route of administration is parenteral (e.g., intratumoral). Incertain embodiments, a preferred route of administration is systemic.

Compositions can be formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified. The preparationof such formulations will be known to those of skill in the art in lightof the present disclosure.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Intratumoral injections are discussed, e.g., in Lammers, et al., “Effectof Intratumoral Injection on the Biodistribution and the TherapeuticPotential of HPMA Copolymer-Based Drug Delivery Systems” Neoplasia.10:788-795 (2006).

Pharmacologically acceptable excipients usable in the rectal compositionas a gel, cream, enema, or rectal suppository, include, withoutlimitation, any one or more of cocoa butter glycerides, syntheticpolymers such as polyvinylpyrrolidone, PEG (like PEG ointments),glycerine, glycerinated gelatin, hydrogenated vegetable oils,poloxamers, mixtures of polyethylene glycols of various molecularweights and fatty acid esters of polyethylene glycol Vaseline, anhydrouslanolin, shark liver oil, sodium saccharinate, menthol, sweet almondoil, sorbitol, sodium benzoate, anoxid SBN, vanilla essential oil,aerosol, parabens in phenoxyethanol, sodium methyl p-oxybenzoate, sodiumpropyl p-oxybenzoate, diethylamine, carbomers, carbopol,methyloxybenzoate, macrogol cetostearyl ether, cocoyl caprylocaprate,isopropyl alcohol, propylene glycol, liquid paraffin, xanthan gum,carboxy-metabisulfite, sodium edetate, sodium benzoate, potassiummetabisulfite, grapefruit seed extract, methyl sulfonyl methane (MSM),lactic acid, glycine, vitamins, such as vitamin A and E and potassiumacetate.

In certain embodiments, suppositories can be prepared by mixing thechemical entities described herein with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum and release the activecompound. In other embodiments, compositions for rectal administrationare in the form of an enema.

In other embodiments, the compounds described herein or a pharmaceuticalcomposition thereof are suitable for local delivery to the digestive orGI tract by way of oral administration (e.g., solid or liquid dosageforms).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the chemicalentity is mixed with one or more pharmaceutically acceptable excipients,such as sodium citrate or dicalcium phosphate and/or: a) fillers orextenders such as starches, lactose, sucrose, glucose, mannitol, andsilicic acid, b) binders such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c)humectants such as glycerol, d) disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate, e) solution retarding agents such asparaffin, f) absorption accelerators such as quaternary ammoniumcompounds, g) wetting agents such as, for example, cetyl alcohol andglycerol monostearate, h) absorbents such as kaolin and bentonite clay,and i) lubricants such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.In the case of capsules, tablets and pills, the dosage form may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like.

In one embodiment, the compositions will take the form of a unit dosageform such as a pill or tablet and thus the composition may contain,along with a chemical entity provided herein, a diluent such as lactose,sucrose, dicalcium phosphate, or the like; a lubricant such as magnesiumstearate or the like; and a binder such as starch, gum acacia,polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or thelike. In another solid dosage form, a powder, marume, solution orsuspension (e.g., in propylene carbonate, vegetable oils, PEGs,poloxamer 124 or triglycerides) is encapsulated in a capsule (gelatin orcellulose base capsule). Unit dosage forms in which one or more chemicalentities provided herein or additional active agents are physicallyseparated are also contemplated; e.g., capsules with granules (ortablets in a capsule) of each drug; two-layer tablets; two-compartmentgel caps, etc. Enteric coated or delayed release oral dosage forms arealso contemplated.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid.

In certain embodiments the excipients are sterile and generally free ofundesirable matter. These compositions can be sterilized byconventional, well-known sterilization techniques. For various oraldosage form excipients such as tablets and capsules sterility is notrequired. The USP/NF standard is usually sufficient.

In certain embodiments, solid oral dosage forms can further include oneor more components that chemically and/or structurally predispose thecomposition for delivery of the chemical entity to the stomach or thelower GI; e.g., the ascending colon and/or transverse colon and/ordistal colon and/or small bowel. Exemplary formulation techniques aredescribed in, e.g., Filipski, K. J., et al., Current Topics in MedicinalChemistry, 2013, 13, 776-802.

Examples include upper-GI targeting techniques, e.g., Accordion Pill(Intec Pharma), floating capsules, and materials capable of adhering tomucosal walls.

Other examples include lower-GI targeting techniques. For targetingvarious regions in the intestinal tract, several enteric/pH-responsivecoatings and excipients are available. These materials are typicallypolymers that are designed to dissolve or erode at specific pH ranges,selected based upon the GI region of desired drug release. Thesematerials also function to protect acid labile drugs from gastric fluidor limit exposure in cases where the active ingredient may be irritatingto the upper GI (e.g., hydroxypropyl methylcellulose phthalate series,Coateric (polyvinyl acetate phthalate), cellulose acetate phthalate,hydroxypropyl methylcellulose acetate succinate, Eudragit series(methacrylic acid-methyl methacrylate copolymers), and Marcoat). Othertechniques include dosage forms that respond to local flora in the GItract, Pressure-controlled colon delivery capsule, and Pulsincap.

Ocular compositions can include, without limitation, one or more of anyof the following: viscogens (e.g., Carboxymethylcellulose, Glycerin,Polyvinylpyrrolidone, Polyethylene glycol); Stabilizers (e.g., Pluronic(triblock copolymers), Cyclodextrins); Preservatives (e.g., Benzalkoniumchloride, ETDA, SofZia (boric acid, propylene glycol, sorbitol, and zincchloride; Alcon Laboratories, Inc.), Purite (stabilized oxychlorocomplex; Allergan, Inc.)).

Topical compositions can include ointments and creams. Ointments aresemisolid preparations that are typically based on petrolatum or otherpetroleum derivatives. Creams containing the selected active agent aretypically viscous liquid or semisolid emulsions, often eitheroil-in-water or water-in-oil. Cream bases are typically water-washable,and contain an oil phase, an emulsifier and an aqueous phase. The oilphase, also sometimes called the “internal” phase, is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic or amphoteric surfactant. As with other carriers or vehicles,an ointment base should be inert, stable, nonirritating andnon-sensitizing.

In any of the foregoing embodiments, pharmaceutical compositionsdescribed herein can include one or more one or more of the following:lipids, interbilayer crosslinked multilamellar vesicles, biodegradeablepoly(D,L-lactic-co-glycolic acid) [PLGA]-based or poly anhydride-basednanoparticles or microparticles, and nanoporous particle-supported lipidbilayers.

Dosages

The dosages may be varied depending on the requirement of the patient,the severity of the condition being treating and the particular compoundbeing employed. Determination of the proper dosage for a particularsituation can be determined by one skilled in the medical arts. Thetotal daily dosage may be divided and administered in portionsthroughout the day or by means providing continuous delivery.

In some embodiments, the compounds described herein are administered ata dosage of from about 0.001 mg/kg to about 500 mg/kg (e.g., from about0.001 mg/kg to about 200 mg/kg; from about 0.01 mg/kg to about 200mg/kg; from about 0.01 mg/kg to about 150 mg/kg; from about 0.01 mg/kgto about 100 mg/kg; from about 0.01 mg/kg to about 50 mg/kg; from about0.01 mg/kg to about 10 mg/kg; from about 0.01 mg/kg to about 5 mg/kg;from about 0.01 mg/kg to about 1 mg/kg; from about 0.01 mg/kg to about0.5 mg/kg; from about 0.01 mg/kg to about 0.1 mg/kg; from about 0.1mg/kg to about 200 mg/kg; from about 0.1 mg/kg to about 150 mg/kg; fromabout 0.1 mg/kg to about 100 mg/kg; from about 0.1 mg/kg to about 50mg/kg; from about 0.1 mg/kg to about 10 mg/kg; from about 0.1 mg/kg toabout 5 mg/kg; from about 0.1 mg/kg to about 1 mg/kg; from about 0.1mg/kg to about 0.5 mg/kg).

Regimens

The foregoing dosages can be administered on a daily basis (e.g., as asingle dose or as two or more divided doses) or non-daily basis (e.g.,every other day, every two days, every three days, once weekly, twiceweeks, once every two weeks, once a month).

In some embodiments, the period of administration of a compounddescribed herein is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, or more. In a furtherembodiment, a period of during which administration is stopped is for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, or more. In an embodiment, a therapeutic compound isadministered to an individual for a period of time followed by aseparate period of time. In another embodiment, a therapeutic compoundis administered for a first period and a second period following thefirst period, with administration stopped during the second period,followed by a third period where administration of the therapeuticcompound is started and then a fourth period following the third periodwhere administration is stopped. In an aspect of this embodiment, theperiod of administration of a therapeutic compound followed by a periodwhere administration is stopped is repeated for a determined orundetermined period of time. In a further embodiment, a period ofadministration is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, or more. In a furtherembodiment, a period of during which administration is stopped is for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, or more.

Methods of Treatment

In some embodiments, methods for treating a subject having condition,disease or disorder in which an increase in NLRP3 signaling may correcta deficiency in innate immune activity (e.g., a condition, disease ordisorder associated with an insufficient immune response) thatcontributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., cancer) are provided.

Indications

In any of the methods described herein, the subject can have a cancer.In some examples of any of the methods described herein, the mammal hasbeen identified as having a cancer, or has been diagnosed as having acancer.

Non-limiting examples of cancer include: acute myeloid leukemia,adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer,appendix cancer, teratoid/rhabdoid tumor, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancer, brain cancer, breast cancer,bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer,chordoma, chronic lymphocytic leukemia, chronic myeloproliferativeneoplasm, colon cancer, colorectal cancer, craniopharyngioma,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, eye cancer, fallopian tube cancer,gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor, germ cell tumor, hairy cell leukemia, head and neckcancer, heart cancer, liver cancer, hypopharngeal cancer, pancreaticcancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia,lip and oral cavity cancer, lung cancer, melanoma, Merkel cellcarcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma,ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer,rectal cancer, salivary gland cancer, skin cancer, small intestinecancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, and vulvarcancer.

In certain embodiments, non-limiting examples of cancer include: breastcancer, colon cancer, rectal cancer, colorectal cancer, pancreaticcancer, and prostate cancer.

Methods for diagnosing a subject as having a cancer or identifying amammal as having a cancer are well known in the art. For example, amedical professional (e.g., a physician, a physician's assistant, or atechnician) can diagnose cancer in a mammal by observing one or moresymptoms of cancer in a mammal. Non-limiting examples of symptoms ofcancer include: fatigue, lump or area of thickening felt under the skin,weight change, jaundice, darkening or redness of the skin, sores thatwon't heal, changes to existing moles, changes in bowel or bladderhabits, persistent cough or trouble breathing, difficulty swallowing,hoarseness, persistent indigestion or discomfort after eating,persistent, unexplained muscle or joint pain, persistent, unexplainedfevers or night sweats, and unexplained bleeding or bruising. Methods ofdiagnosing a subject as having a cancer or identifying a subject ashaving a cancer can further include performing one or more diagnostictests (e.g., performing one or more diagnostic tests on a biopsy or ablood sample).

In some examples of any of the methods described herein, a subject canbe a subject having a cancer, a subject diagnosed as having a cancer, ora subject identified as having a cancer that has been unresponsive to apreviously administered treatment for cancer. Diagnostic tests fordiagnosing a subject as having a cancer or identifying a mammal ashaving a cancer are known in the art.

In some embodiments, methods for treating a subject having condition,disease or disorder in which an increase in NLRP3 signaling may correcta deficiency in innate immune activity (e.g., a condition, disease ordisorder associated with an insufficient immune response) thatcontributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., cancer) are provided.

In some embodiments, the present invention provides a method of treatingcancer, wherein the cancer can be any cancer that does not elicit anoptimal innate immune system response.

Innate immune system refers to a part of the immune system consisting ofcells that react to threats for the organism like infections or cancerin an antigen-non-specific way and stimulate the adaptive,antigen-specific immune system. In general, complete removal of thethreat and long-lasting protection (=immunity) requires activity of theadaptive, antigen-specific immune system that in turn depends onstimulation by the innate immune system.

In some embodiments, the present invention provides a method of treatingcase, the cancer is selected based on resistance to T-cell checkpointinhibition, either independent of cancer type and based on failure torespond to previous T-cell checkpoint inhibitor therapy or based oncancer type that is generally resistant to T-cell checkpoint inhibitortherapy such as hormone receptor positive breast cancer, microsatellitestable colon or rectal cancer, pancreatic cancer and prostate cancer.

In certain other embodiments, the present invention provides a method oftreating cancer comprising an NLPR3 agonist of the present invention totreat non-inflamed tumors with low CD8+ T-cell infiltration to enhancetumor immunogenicity and promote inflammatory responses. For example,the combination may be used to treat a solid tumor based on results of abiopsy that demonstrated low CD8+ T-cell infiltration or low expressionof genes produced by CD8+ T-cells.

Resistance to T-cell checkpoint inhibition refers to cancer progressionon therapy or lack of response within 6 months of therapy according toconsensus response criteria for the respective cancer, such as RECIST1.1for most solid tumors.

T-cell infiltration refers to percent of T-cells of all nucleated cellsby immunohistochemistry of tumor biopsy specimens.

CD8+ T-cell infiltration refers to percent of CD8+ cells of allnucleated cells by immunohistochemistry of tumor biopsy specimens.

In addition to immunohistochemistry for quantifying CD8+ T-cells inbiopsy specimens, expression of genes produced by CD8+ T-cells likeinterferon-γ can be measured by quantifying mRNA using for example nextgeneration sequencing and inform about CD8+ T-cell infiltration.Thresholds for low and high CD8+ T-cell infiltration byimmunohistochemistry of mRNA quantifying techniques are being developedby various groups and take the spectrum of CD8+ T-cell infiltrationacross cancers as well as for specific cancers into account.

In any of the methods described herein, the subject can have aninfectious disease. In some examples of any of the methods describedherein, the subject has been identified as having an infectious disease,or has been diagnosed as having an infectious disease. For example, aninfectious disease can be caused by a bacterium, virus, fungus,parasite, or a mycobacterium.

Non-limiting examples of infectious disease include: Acinobacterinfection, actinomycosis, African sleeping sickness, acquiredimmunodeficiency syndrome, amebiasis, anaplasmosis, anthrax,Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever,ascariasis, aspergillosis, astrovirus infection, babesiosis, Bacilluscereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroidesinfection, balantidiasis, Baylisascaris infection, BK virus infection,black piedra, Blastocystic hominis infection, blastomycosis, Bolivianhemorrhagic fever, botulism, Brazilian hemorrhagic fever, brucellosis,bubonic plaque, Burkholderi infection, Buruli ulcer, Calicivirusinfection, camptobacteriosis, candidiasis, cat-scratch disease,cellulitis, Chagas disease, chancroid, chickenpox, chikungunya,chlamydia, Chlamydophila pneumoniae infection, cholera,chromoblastomycosis, clonorchiasis, Clostridium difficile infection,coccidioidomycosis, Colorado tick fever, common cold, Creutzfeldt-Jakobdisease, Crimean-Congo hemorrhagic fever, crytococcosis,cryptosporidiosis, cutaneous larva migrans, cyclosporiasis,cysticercosis, cytomegalovirus infection, dengue fever, Desmodesmusinfection, deintamoebiasis, diphtheria, diphyllobothriasis,dracunculiasis, ebola hemorrhagic fever, echinococcosis, ehrlichiosis,enterobiasis, Enterococcus infection, Enterovirus infection, epidemictyphus, erythema infection, exanthema subitum, fasciolopsiasis,fasciolosis, fatal familial insomnia, filariasis, food poisoning byClostridium myonecrosis, free-living amebic infection, Fusobacteriuminfection, gas gangrene, geotrichosis, Gerstmann-Straussler-Scheinkersyndrome, giardiasis, glanders, gnathostomiasis, gonorrhea, granulomainguinale, Group A streptococcal infection, Group B streptococcalinfection, Haemophilus influenzae infection, hand foot and mouthdisease, hantavirus pulmonary syndrome, Heartland virus disease,Heliobacter pylori infection, hemolytic-uremic syndrome, hemorrhagicfever with renal syndrome, hepatitis A, hepatitis B, hepatitis C,hepatitis D, hepatitis E, herpes simplex, histoplasmosis, hookworminfection, human bocavirus infection, human ewingii ehrlichiosis, humangranulocyte anaplasmosis, human metapneuomovirus infection, humanmonocytic ehrlichiosis, human papillomavirus infection, humanparainfluenza virus infection, hymenolepiasis, Epstein-Barr virusinfectious mononucleosis, influenza, isosporiasis, Kawasaki disease,keratitis, Kingella kingae infection, kuru, lassa fever, Legionnaires'disease, Pontiac fever, leishmaniasis, leprosy, leptospirosis,listeriosis, lyme disease, lymphatic filariasis, lymphocyticchoriomeningitis, malaria, Marburg hemorrhagic fever, measles, MiddleEast respiratory syndrome, melioidosis, meningitis, meningococcaldisease, metagonimiasis, microsporidiosis, molluscum contagiosum,monkeypox, mumps, murine typhus, mycoplasma pneumonia, mycetoma,myiasis, neonatal conjunctivitis, variant Creutzfeldt-Jakob disease,nocardiosis, onchocerciasis, paracoccidioidomycosis, paragonimiasis,pasteurellosis, pediculosis capitis, pediculosis corporis, pediculosispubis, pelvic inflammatory disease, pertussis, plague, pneumonia,poliomyelitis, Prevotella infection, primary amoebicmeningoencephalitis, progressive multifocal leukoencephalopathy,psittacosis, Q fever, rabies, relapsing fever, respiratory syncytialvirus infection, rhinosporidiosis, rhinovirus infection, rickettsialinfection, rickettsialpox, Rift Valley Fever, Rocky Mountain spottedfever, rotavirus infection, rubella, salmonellosis, severe acuterespiratory syndrome, scabies, schistosomiasis, sepsis, shigellosis,shingles, smallpox, sporothrichosis, staphylococcal food poisoning,staphylococcal infection, strongyloidiasis, subacute sclerosingpanencephalitis, syphilis, taeniasis, tetanus, tinea barabe, tineacapitis, tinea corporis, tinea cruris, tinea manum, tinea nigra, tineapedis, tinea unguium, tinea versicolor, toxocariasis, trachoma,toxoplasmosis, trichinosis, trichomoniasis, trichuriasis, tuberculosis,tularemia, typhoid fever, Ureaplasma urealyticum infection, valleyfever, Venezuelan hemorrhagic fever, viral pneumonia, West Nile fever,white piedra, Yersiniapsuedotuberculosis infection, yersiniosis, yellowfever, and zygomycosis.

Methods for diagnosing a subject as having an infectious disease, oridentifying a subject as having an infectious disease are well known inthe art. For example, a medical professional (e.g., a physician, aphysician's assistant, or a technician) can diagnose infectious diseasein a subject by observing one or more symptoms of infectious disease ina subject. Non-limiting examples of symptoms of infectious diseaseinclude: fever, diarrhea, fatigue, and muscle aches. Methods ofdiagnosing a mammal as having an infectious disease or identifying asubject as having an infectious disease can further include performingone or more diagnostic tests (e.g., performing one or more diagnostictests on a biopsy or a blood sample). Diagnostic tests for diagnosing asubject as having an infectious disease or identifying a subject ashaving an infectious disease are known in the art.

Combination Therapy

This disclosure contemplates both monotherapy regimens as well ascombination therapy regimens.

In some embodiments, the methods described herein can further includeadministering one or more additional therapies (e.g., one or moreadditional therapeutic agents and/or one or more therapeutic regimens)in combination with administration of the compounds described herein.

In certain embodiments, the methods described herein can further includeadministering one or more additional cancer therapies.

The one or more additional cancer therapies can include, withoutlimitation, surgery, radiotherapy, chemotherapy, toxin therapy,immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine,hepatitis B vaccine, Oncophage, Provenge) and gene therapy, as well ascombinations thereof. Immunotherapy, including, without limitation,adoptive cell therapy, the derivation of stem cells and/or dendriticcells, blood transfusions, lavages, and/or other treatments, including,without limitation, freezing a tumor.

In some embodiments, the one or more additional cancer therapies ischemotherapy, which can include administering one or more additionalchemotherapeutic agents.

In certain embodiments, the additional cancer therapy comprises(chemotherapeutic agent) an immunomodulatory moiety, e.g., an immunecheckpoint inhibitor. In certain of these embodiments, the immunecheckpoint inhibitor targets an immune checkpoint receptor selected fromCTLA-4, PD-1, PD-L1, PD-1—PD-L1, PD-1—PD-L2, T cell immunoglobulin andmucin 3 (TIM3 or HAVCR2), Galectin 9—TIM3, Phosphatidylserine—TIM3,lymphocyte activation gene 3 protein (LAG3), MHC class II—LAG3,4-1BB-4-1BB ligand, OX40-OX40 ligand, GITR, GITR ligand—GITR, CD27,CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40 ligand,HVEM-LIGHT-LTA, HVEM, HVEM—BTLA, HVEM—CD160, HVEM—LIGHT,HVEM-BTLA-CD160, CD80, CD80—PDL-1, PDL2—CD80, CD244, CD48—CD244, CD244,ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2,Butyrophilins, including BTNL2, Siglec family, TIGIT and PVR familymembers, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICA and MICB, CD244,CD28, CD86—CD28, CD86—CTLA, CD80—CD28, Phosphatidylserine, TIM3,Phosphatidylserine—TIM3, SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, andCD155 (e.g., CTLA-4 or PD1 or PD-L1) and other immunomodulatory agents,such as interleukin-2 (IL-2), indoleamine 2,3-dioxygenase (IDO), IL-10,transforming growth factor-β (TGFβ), CD39, CD73 Adenosine-CD39-CD73, andCXCR4-CXCL12. See, e.g., Postow, M. J. Clin. Oncol. 33, 1 (2015).

In certain embodiments, the immune checkpoint inhibitor targets animmune checkpoint receptor selected from CTLA-4, PD-1, PD-L1,PD-1—PD-L1, and PD-1-PD-L2.

In certain embodiments, the immune checkpoint inhibitor is selectedfrom: nivolumab (also known as “OPDIVO”; formerly designated 5C4,BMS-936558, MDX-1106, or ONO-4538), pembrolizumab (also known as“KEYTRUDA”, lambrolizumab, and MK-3475. See WO 2008/156712), PDR001(Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; AMP-514; see WO2012/145493), cemiplimab (REGN-2810) (Regeneron; see WO 2015/112800),JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J Hematol. Oncol.10:136 (2017)), BGB-A317 (Beigene; see WO 2015/35606 and US2015/0079109), INCSHR1210 (SHR-1210; Jiangsu Hengrui Medicine; see WO2015/085847; Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)),TSR-042 (ANB011; Tesaro Biopharmaceutical; see WO2014/179664), GLS-010(WBP3055; Wuxi/Harbin Gloria Pharmaceuticals; see Si-Yang Liu et al., J.Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (SorrentoTherapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO2017/040790), MGD013 (Macrogenics); IBI308 (Innovent; see WO2017/024465, WO 2017/025016, WO 2017/132825, WO2017/133540); BMS-936559(formerly 12A4 or MDX-1105; see, e.g., U.S. Pat. No. 7,943,743 and WO2013/173223), MPDL3280A (also known as RG7446, atezolizumab, andTECENTRIQ; U.S. Pat. No. 8,217,149; see, also, Herbst et al. (2013) JClin Oncol 31(suppl):3000), durvalumab (IMFINZI; MEDI-4736; AstraZeneca;see WO 2011/066389), avelumab (Pfizer; MSB-0010718C; BAVENCIO; see WO2013/079174), STI-1014 (Sorrento; see WO2013/181634), CX-072 (Cytomx;see WO2016/149201), KN035 (3D Med/Alphamab; see Zhang et al.,CellDiscov. 7:3 (March 2017), LY3300054 (Eli Lilly Co.; see, e.g, WO2017/034916), CK-301 (Checkpoint Therapeutics; see Gorelik et al.,AACR:Abstract 4606 (April 2016)); urelumab, PF-05082566, MEDI6469,TRX518, varlilumab, CP-870893, BMS-986016, MGA271, lirilumab, IPH2201,emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, Bavituximab,CC-90002, bevacizumab, MNRP1685A, ipilimumab (YERVOY; U.S. Pat. No.6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; WO 2016/196237),and tremelimumab (formerly ticilimumab, CP-675,206; AstraZeneca; see,e.g., WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39(2007)).

In certain embodiments, the immune checkpoint inhibitor is selectedfrom: nivolumab, pembrolizumab, JS001, BGB-A317, INCSHR1210, TSR-042,GLS-010, STI-1110, MGD013, IBI308, BMS-936559, atezolizumab, durvalumab,avelumab, STI-1014, CX-072, KN035, LY3300054, CK-301, urelumab,PF-05082566, MEDI6469, TRX518, varlilumab, BMS-986016, ipilimumab,AGEN-1884, and tremelimumab.

In certain of these embodiments, the immune checkpoint inhibitor isselected from: Urelumab, PF-05082566, MEDI6469, TRX518, Varlilumab,CP-870893, Pembrolizumab (PD1), Nivolumab (PD1), Atezolizumab (formerlyMPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab (PD-L1), PDR001 (PD1),BMS-986016, MGA271, Lirilumab, IPH2201, Emactuzumab, INCB024360,Galunisertib, Ulocuplumab, BKT140, Bavituximab, CC-90002, bevacizumab,and MNRP1685A.

In certain embodiments, the immune checkpoint inhibitor is selectedfrom: nivolumab, ipilimumab, pembrolizumab, atezolizumab, durvalumab andavelumab.

In certain embodiments, the immune checkpoint inhibitor is selectedfrom: nivolumab and ipilimumab.

In certain embodiments, the additional anti-cancer agent(chemotherapeutic agent) is a STING agonist. For example, the STINGagonist can include cyclic di-nucleotides, such as cAMP, cGMP, and cGAMPas well as modified cyclic di-nucleotides that include one or more ofthe following modification features (2′-O/3′-0 linkage, phosphorothioatelinkage, adenine and/or guanine analogue, 2′-OH modification (e.g.,—OCH₃ or replacement, e.g., —F or N3). See, e.g., WO 2014/189805.

In certain embodiments, the additional chemotherapeutic agent is analkylating agent. Alkylating agents are so named because of theirability to alkylate many nucleophilic functional groups under conditionspresent in cells, including, but not limited to cancer cells. In afurther embodiment, an alkylating agent includes, but is not limited to,Cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide and/or oxaliplatin. In an embodiment, alkylating agents canfunction by impairing cell function by forming covalent bonds with theamino, carboxyl, sulfhydryl, and phosphate groups in biologicallyimportant molecules or they can work by modifying a cell's DNA. In afurther embodiment an alkylating agent is a synthetic, semisynthetic orderivative.

In certain embodiments, the additional chemotherapeutic agent is ananti-metabolite. Anti-metabolites masquerade as purines or pyrimidines,the building-blocks of DNA and in general, prevent these substances frombecoming incorporated in to DNA during the “S” phase (of the cellcycle), stopping normal development and division. Anti-metabolites canalso affect RNA synthesis. In an embodiment, an antimetabolite includes,but is not limited to azathioprine and/or mercaptopurine. In a furtherembodiment an anti-metabolite is a synthetic, semisynthetic orderivative.

In certain embodiments, the additional chemotherapeutic agent is a plantalkaloid and/or terpenoid. These alkaloids are derived from plants andblock cell division by, in general, preventing microtubule function. Inan embodiment, a plant alkaloid and/or terpenoid is a vinca alkaloid, apodophyllotoxin and/or a taxane. Vinca alkaloids, in general, bind tospecific sites on tubulin, inhibiting the assembly of tubulin intomicrotubules, generally during the M phase of the cell cycle. In anembodiment, a vinca alkaloid is derived, without limitation, from theMadagascar periwinkle, Catharanthus roseus (formerly known as Vincarosea). In an embodiment, a vinca alkaloid includes, without limitation,Vincristine, Vinblastine, Vinorelbine and/or Vindesine. In anembodiment, a taxane includes, but is not limited, to Taxol, Paclitaxeland/or Docetaxel. In a further embodiment a plant alkaloid or terpernoidis a synthetic, semisynthetic or derivative. In a further embodiment, apodophyllotoxin is, without limitation, an etoposide and/or teniposide.In an embodiment, a taxane is, without limitation, docetaxel and/orortataxel. In an embodiment, a cancer therapeutic is a topoisomerase.Topoisomerases are essential enzymes that maintain the topology of DNA.Inhibition of type I or type II topoisomerases interferes with bothtranscription and replication of DNA by upsetting proper DNAsupercoiling. In a further embodiment, a topoisomerase is, withoutlimitation, a type I topoisomerase inhibitor or a type II topoisomeraseinhibitor. In an embodiment a type I topoisomerase inhibitor is, withoutlimitation, a camptothecin. In another embodiment, a camptothecin is,without limitation, exatecan, irinotecan, lurtotecan, topotecan, BNP1350, CKD 602, DB 67 (AR67) and/or ST 1481. In an embodiment, a type IItopoisomerase inhibitor is, without limitation, epipodophyllotoxin. In afurther embodiment an epipodophyllotoxin is, without limitation, anamsacrine, etoposid, etoposide phosphate and/or teniposide. In a furtherembodiment a topoisomerase is a synthetic, semisynthetic or derivative,including those found in nature such as, without limitation,epipodophyllotoxins, substances naturally occurring in the root ofAmerican Mayapple (Podophyllum peltatum).

In certain embodiments, the additional chemotherapeutic agent is astilbenoid. In a further embodiment, a stilbenoid includes, but is notlimited to, Resveratrol, Piceatannol, Pinosylvin, Pterostilbene,Alpha-Viniferin, Ampelopsin A, Ampelopsin E, Diptoindonesin C,Diptoindonesin F, Epsilon-Vinferin, Flexuosol A, Gnetin H, HemsleyanolD, Hopeaphenol, Trans-Diptoindonesin B, Astringin, Piceid andDiptoindonesin A. In a further embodiment a stilbenoid is a synthetic,semisynthetic or derivative.

In certain embodiments, the additional chemotherapeutic agent is acytotoxic antibiotic. In an embodiment, a cytotoxic antibiotic is,without limitation, an actinomycin, an anthracenedione, ananthracycline, thalidomide, dichloroacetic acid, nicotinic acid,2-deoxyglucose and/or chlofazimine. In an embodiment, an actinomycin is,without limitation, actinomycin D, bacitracin, colistin (polymyxin E)and/or polymyxin B. In another embodiment, an antracenedione is, withoutlimitation, mitoxantrone and/or pixantrone. In a further embodiment, ananthracycline is, without limitation, bleomycin, doxorubicin(Adriamycin), daunorubicin (daunomycin), epirubicin, idarubicin,mitomycin, plicamycin and/or valrubicin. In a further embodiment acytotoxic antibiotic is a synthetic, semisynthetic or derivative.

In certain embodiments, the additional chemotherapeutic agent isselected from endostatin, angiogenin, angiostatin, chemokines,angioarrestin, angiostatin (plasminogen fragment), basement-membranecollagen-derived anti-angiogenic factors (tumstatin, canstatin, orarrestin), anti-angiogenic antithrombin III, signal transductioninhibitors, cartilage-derived inhibitor (CDI), CD59 complement fragment,fibronectin fragment, gro-beta, heparinases, heparin hexasaccharidefragment, human chorionic gonadotropin (hCG), interferonalpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12,kringle 5 (plasminogen fragment), metalloproteinase inhibitors (TIMPs),2-methoxyestradiol, placental ribonuclease inhibitor, plasminogenactivator inhibitor, platelet factor-4 (PF4), prolactin 16 kD fragment,proliferin-related protein (PRP), various retinoids,tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growthfactor-beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment)and the like.

In certain embodiments, the additional chemotherapeutic agent isselected from abiraterone acetate, altretamine, anhydrovinblastine,auristatin, bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide,cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide,hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine,lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard),melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin,mitomycin, methotrexate, taxanes, nilutamide, onapristone, paclitaxel,prednimustine, procarbazine, RPR109881, stramustine phosphate,tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine,vindesine sulfate, and vinflunine.

In certain embodiments, the additional chemotherapeutic agent isplatinum, cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, azathioprine, mercaptopurine,vincristine, vinblastine, vinorelbine, vindesine, etoposide andteniposide, paclitaxel, docetaxel, irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate, teniposide, 5-fluorouracil, leucovorin,methotrexate, gemcitabine, taxane, leucovorin, mitomycin C,tegafur-uracil, idarubicin, fludarabine, mitoxantrone, ifosfamide anddoxorubicin. Additional agents include inhibitors of mTOR (mammaliantarget of rapamycin), including but not limited to rapamycin,everolimus, temsirolimus and deforolimus.

In still other embodiments, the additional chemotherapeutic agent can beselected from those delineated in U.S. Pat. No. 7,927,613.

In yet another embodiment, the methods can further include administeringone or both of: (i) one or more anti-fungal agents (e.g., selected fromthe group of bifonazole, butoconazole, clotrimazole, econazole,ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole,sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole,epoziconazole, fluconazole, isavuconazole, itraconazole, posaconazole,propiconazole, ravusconazole, terconazole, voriconazole, abafungin,amorolfin, butenafine, naftifine, terbinafine, anidulafungin,caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine,5-fluorocytosine, griseofulvin, haloprogin, tolnaflate, undecylenicacid, and balsam of peru) and (ii) one or more antibiotics (e.g.,selected from the group of amikacin, gentamicin, kanamycin, neomycin,netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin,geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem,imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin,cefalothin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil,cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin,telavancin, dalbavancin, oritavancin, clindamycin, lincomycin,daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam,furazolidone, nitrofurantoin, linezolid, posizolid, radezolid,torezolid, amoxicillin, ampicillin, azlocillin, carbenicillin,cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin,nafcillin, oxacillin, penicillin G, penicillin V, piperacillin,penicillin G, temocillin, ticarcillin, amoxicillin, calvulanate,ampicillin, subbactam, piperacillin, tazobactam, ticarcillin,clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine,silver sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfanilimide,sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole,sulfonamideochrysoidine, demeclocycline, minocycline, oytetracycline,tetracycline, clofazimine, dapsone, dapreomycin, cycloserine,ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin,rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin,fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin,dalopristin, thiamphenicol, tigecycyline, tinidazole, trimethoprim, andteixobactin).

In certain embodiments, the second therapeutic agent or regimen isadministered to the subject prior to contacting with or administeringthe chemical entity (e.g., about one hour prior, or about 6 hours prior,or about 12 hours prior, or about 24 hours prior, or about 48 hoursprior, or about 1 week prior, or about 1 month prior).

In other embodiments, the second therapeutic agent or regimen isadministered to the subject at about the same time as contacting with oradministering the chemical entity. By way of example, the secondtherapeutic agent or regimen and the chemical entity are provided to thesubject simultaneously in the same dosage form. As another example, thesecond therapeutic agent or regimen and the chemical entity are providedto the subject concurrently in separate dosage forms.

In still other embodiments, the second therapeutic agent or regimen isadministered to the subject after contacting with or administering thechemical entity (e.g., about one hour after, or about 6 hours after, orabout 12 hours after, or about 24 hours after, or about 48 hours after,or about 1 week after, or about 1 month after).

Patient Selection

In some embodiments, the methods described herein further include thestep of identifying a subject (e.g., a patient) in need of suchtreatment (e.g., by way of biopsy, endoscopy, or other conventionalmethod known in the art). In certain embodiments, the NLRP3 protein canserve as a biomarker for certain types of cancer.

In some embodiments, the chemical entities, methods, and compositionsdescribed herein can be administered to certain treatment-resistantpatient populations (e.g., patients resistant to checkpoint inhibitors).

In some embodiments, the compounds of the present invention may be usedin therapy. In certain embodiments, the present invention provides acombined preparation of a compound of the present invention, or apharmaceutically acceptable salt thereof, and additional therapeuticagent(s) for simultaneous, separate or sequential use in therapy.

In some embodiments, a compound of the present invention, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition containing the same, may be used as a medicament. In certainembodiments, the compounds of the invention may be used for themanufacture of a medicament for the treatment of cancer.

In certain embodiments, the compounds of the invention may be used forthe manufacture of a medicament for modulating NLRP3 activity. Incertain embodiments, the modulating comprises agonizing NLRP3.

Methods of Preparation

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. For example, the compounds described hereincan be synthesized, e.g., using one or more of the methods describedherein and/or using methods described in, e.g., US 2015/0056224.

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art and include, for example, thosesuch as described in Larock, R. C., Comprehensive OrganicTransformations, 2^(nd) Edition, Wiley-VCH, New York, NY (1999); Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 5th Edition,Wiley (2014); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof. The starting materials used inpreparing the compounds of the invention are known, made by knownmethods, or are commercially available. The skilled artisan will alsorecognize that conditions and reagents described herein that can beinterchanged with alternative art-recognized equivalents. For example,in many reactions, triethylamine can be interchanged with other bases,such as non-nucleophilic bases (e.g. diisopropylamine,1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine, ortetrabutylphosphazene).

The skilled artisan will recognize a variety of analytical methods thatcan be used to characterize the compounds described herein, including,for example, ¹H NMR, heteronuclear NMIR, mass spectrometry, liquidchromatography, and infrared spectroscopy. The foregoing list is asubset of characterization methods available to a skilled artisan and isnot intended to be limiting.

The following abbreviations have the indicated meanings:

-   -   ACN=acetonitrile    -   AcOH=acetic acid    -   BOP=(Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium        hexafluorophosphate    -   CDCl₃=chloroform-d    -   CD₃OD=methanol-d₄    -   CH₂Cl₂=dichloromethane    -   CH₃ReO₃=methyltrioxorhenium    -   Cs₂CO₃=cesium carbonate    -   CuI=copper (I) iodide    -   d=doublet    -   DBU=1,8-diazabicycloundec-7-ene    -   DCM=dichloromethane    -   DIEA=N,N-diisopropylethylamine    -   DMF=N,N-dimethylformamide    -   DMSO=dimethylsulfoxide    -   ES=electrospray ionization    -   Et₂O=diethyl ether    -   EtOAc=ethyl acetate    -   EtOH=ethanol    -   equiv=equivalents    -   g=gram(s)    -   h=hour(s)    -   HCl=hydrogen chloride (usually as a solution)    -   H₂O=water    -   H₂O₂=hydrogen peroxide    -   HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxide hexafluorophosphate    -   HPLC=high-performance liquid chromatography    -   Hunig's base=N,N-Diisopropylethylamine    -   I₂=iodine    -   K₂CO₃=potassium carbonate    -   K₂HPO₄=potassium phosphate, dibasic    -   KI=potassium iodide    -   kg=kilogram(s)    -   LC/MS=liquid chromatography mass spectrometer    -   LiBH₄=lithium borohydride    -   m=multiplet    -   m/z=mass to charge ratio    -   M=molar    -   m-CPBA=meta-chloroperoxybenzoic acid    -   mg=milligram(s)    -   MeOH=methanol    -   MHz=megahertz    -   mL=milliliter(s)    -   mmol=millimole(s)    -   min=minute(s)    -   NaHCO₃=sodium bicarbonate    -   Na₂CO₃=sodium carbonate    -   NaOH=sodium hydroxide    -   Na₂SO₄=sodium sulfate    -   NEt₃ and TEA=triethylamine    -   NH₄OH or NH₃H₂O=ammonium hydroxide    -   NH₄HCO₃=ammonium bicarbonate    -   nm=nanometer    -   PdCl₂(PPh₃)₂=bis(triphenylphosphine)palladium (II) dichloride    -   Pd(dppf)Cl₂=[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)    -   Pd(dppf)Cl₂DCM=[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)        dichloromethane complex    -   Pd(OH)₂=palladium hydroxide    -   PMB=para-methoxybenzyl    -   POCl₃=phosphorous oxychloride    -   ppm=parts per million    -   Pt=platinum    -   Pt/C=platinum on carbon    -   s=singlet    -   t=triplet    -   T3P=n-propylphosphonic anhydride    -   TFA=trifluoroacetic acid    -   TLC=thin layer chromatography    -   TsCl=para-toluenesulfonyl chloride    -   ° C.=degrees Celsius    -   μmol=micromole(s)

The compounds of the present invention can be prepared in a number ofways well known to one skilled in the art of organic synthesis. Thecompounds of the present invention can be synthesized using the methodsdescribed below, together with synthetic methods known in the art ofsynthetic organic chemistry, or variations thereon as appreciated bythose skilled in the art. Preferred methods include, but are not limitedto, those described below.

The compounds of this invention may be prepared using the reactions andtechniques described in this section. The reactions are performed insolvents appropriate to the reagents and materials employed and aresuitable for the transformations being effected. Also, in thedescription of the synthetic methods described below, it is to beunderstood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment and work up procedures, are chosen to be the conditionsstandard for that reaction, which should be readily recognized by oneskilled in the art. It is understood by one skilled in the art oforganic synthesis that the functionality present on various portions ofthe molecule must be compatible with the reagents and reactionsproposed. Such restrictions to the substituents that are compatible withthe reaction conditions will be readily apparent to one skilled in theart and alternate methods must then be used. This will sometimes requirea judgment to modify the order of the synthetic steps or to select oneparticular process scheme over another in order to obtain a desiredcompound of the invention. It will also be recognized that another majorconsideration in the planning of any synthetic route in this field isthe judicious choice of the protecting group used for protection of thereactive functional groups present in the compounds described in thisinvention.

The compounds of this invention may be prepared using the reactions andtechniques described in this section and the accompanying Schemes.

The synthesis of the compounds of Formula (I) can be effected using themethods summarized in Scheme 1.

Step 1: The first step of Scheme 1 begins with a suitably functionalizedquinolinol (i). If desired, the groups R⁹, R¹⁰, R¹¹, and R¹² may be thegroups R², R³, R⁴, and R⁵ found in the compound of Formula (I).Alternatively, one or more of these groups may be groups that can bemodified at a later stage of the synthesis, such as bromo. Thisquinolinol may be purchased commercially or may be synthesized bymethods known to one skilled in the art. In step 1 of scheme 1, thenitration product (ii) may prepared by treating compound (i) with asuitable nitrating reagent, such as nitric acid, in a suitable solvent,such as propionic acid, at an appropriate temperature, such as 120° C.

Step 2: In step 2 of Scheme 1, the alcohol group of compound (ii) may betransformed into a halogen group or sulfonate ester, such as chloro,bromo, or triflate. If the desired group Y is chloro, thistransformation may be accomplished by treating compound (ii) with areagent such as phosphoryl chloride in a solvent such as DMF at anappropriate temperature, such as 70° C. Alternatively, if the desiredgroup Y is bromo, this transformation may be accomplished by treatingcompound (ii) with a reagent such as phosphorous tribromide in a solventsuch as DMF. Alternatively, if the desired group Y is triflate, thistransformation may be effected by treating compound (ii) with a reagentsuch as trifluoromethanesulfonyl chloride, a reagent such as4-dimethylaminopyridine, and a base such as Hunig's base in a solventsuch as dichloromethane.

Step 3: In step 3 of Scheme 1, the halogen Y of compound (iii) may betransformed into nitrogen-R¹³ group of compound (iv). The group R¹³ maybe the group R¹ desired in the final compound; alternatively, it can bea group that may be transformed into group R¹ at a later stage of thesynthesis. One skilled in the art will recognize that the means toeffect this transformation will depend on the nature of the nitrogen-R¹³group and Y. For example, if Y is chloro, this transformation may beeffected by heating compound (iii) to a suitable temperature, such as40° C., with a suitably substituted amine, or ammonium hydroxide, in anappropriate solvent, such as acetonitrile.

Step 4: In step 4 of scheme 1, the nitro group of compound (iv) may betransformed to amine (v) with a suitable reducing agent, such as tin(II)chloride-dihydrate, in a suitable solvent, such as ethyl acetate, at anappropriate temperature, such as refluxing.

Step 5: In step 5 of Scheme 1, the Z—R¹⁴ group may be group R⁶.Alternatively, the R¹⁴ substitution on the Z group may be a protectinggroup, such as a Boc or Cbz, that would allow for further modificationat a later step. Step 5 may be accomplished by treating compound (v)with a suitably functionalized carboxylic acid, a coupling agent, suchas 1-propanephosphonic anhydride, in the presence of a base, such aspyridine, in a suitable solvent such as ethyl acetate or DMF, at anappropriate temperature, such as room temperature to provide compound(vi).

Step 6: In step 6 of Scheme 1, compound (vi) may be treated with ahydroxide base, such as sodium hydroxide, in a suitable solvent, such asethanol, at an appropriate temperature, such as 80° C., to give cyclizedcompound (vii).

Step 7: In step 7 of Scheme 1, compound (vii) may be transformed intoN-oxide (viii) by treatment with an appropriate oxidant, such asmeta-chloroperoxybenzoic acid, in an appropriate solvent, such as DCM.

Step 8: In step 8 of Scheme 1, compound (viii) may be transformed intoamine (ix) by treatment with an appropriate activating reagent, such astosyl chloride, and a source of ammonia, such as ammonium hydroxide or amixture of ammonium chloride and triethylamine, in an appropriatesolvent, such as DCM.

Step 9: Step 9 of Scheme 1 is a step or series of steps to transform thegroup R¹¹ in compound (ix) to the group R⁴ found in molecule (x). Forexample, if R¹¹ is bromo and the desired group R⁴ is an aromatic orheteroaromatic group, this transformation may be effected by reactingcompound (ix) with an optionally protected aromatic or heteroaromaticboronic acid or boronic ester, a catalyst such as PdCl₂(dppf)-DCMcomplex, and a base such as tripotassium phosphate, in an appropriatesolvent mixture, such as dioxane and water. If the group installedcontains a protecting group, a further optional step may be conducted toremove that protecting group under appropriate conditions, if desired.For example, if the group installed was a pyrazole with atetrahydropyran protecting group, the tetrahydropyran may be removed byreaction with an acid such as trifluoroacetic acid in a solvent such asdichloromethane. Alternatively, if R¹¹ is bromo and the desired group R⁴is an aromatic or heteroaromatic group, this transformation may beeffected by reacting compound (ix) first with a compound such asbis(pinacolato)diboron, a reagent such as potassium acetate, and acatalyst such as PdCl₂(dppf)-DCM complex in a solvent such as dioxane,then reacting the resulting boronic ester with an appropriate aryl orheteroaryl halide, a base such as sodium carbonate, and a catalyst suchas tetrakis(triphenylphosphine)palladium(0) in an appropriate solventmixture, such as dioxane and water. Alternatively, if R¹¹ is bromo andthe desired group R⁴ is a heterocycle linked through a nitrogen atom,this step may be effected by reaction of compound (ix) with theappropriate heterocycle in the presence of a copper source such ascopper (I) iodide, a base such as sodium carbonate, and a ligand such asN,N′-dimethylethane-1,2-diamine in an appropriate solvent, such as DMSO.

Step 10: Step 10 of Scheme 1 is an optional step in which R¹⁴ is aprotecting group such as a Boc or Cbz group. This transformation may beaccomplished with the treatment of compound (x) with an acid, such astrifluoroacetic acid (neat) or hydrochloric acid in an appropriatesolvent, such as dioxane, to give compound (xi).

Step 11: Step 11 of Scheme 1 is an optional step in which the Z—H groupof compound (xi), may be transformed to the R⁶ group in compound (xii),if further modification is desired. This transformation may beaccomplished by treating compound (xi) with a suitable fuctionalizedcarboxylic acid, in the presence of a suitable base such as Hunig'sbase, with a suitable coupling agent, such as 1-propanephosphonicanhydride, in a suitable solvent, such as DMF, at an appropriatetemperature, such as room temperature.

Step 12: Step 12 of Scheme 1 is an optional step or series of steps totransform the groups R⁹, R¹⁰, R¹², and R¹³ in compound (xii) to thegroup R², R³, R⁵, and R¹ found in the compound of Formula (I).

One skilled in the art will recognize that a number of these steps maybe performed in alternative order, depending on the groups desired inthe compound of Formula (I). For example, for some molecules, thetransformation of the group R¹¹ to R⁴ described in Step 9 may beconducted after to the transformation of the group Z—R¹⁴ to the group R⁶described in Step 11.

Evaluation of Biological Activity Measurement of IL-1β Production inPMA-Differentiated THP-1 Cells

THP-1 cells were purchased from the American Type Culture Collection andsub-cultured according to instructions from the supplier. Prior toexperiments, cells were cultured in RPMI 1640 containing 10% heatinactivated FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml),and maintained in log phase prior to experimental setup. Prior to theexperiment THIP-1 were treated with PMA (Phorbol 12-myristate13-acetate) (10 μg/ml) for 24 hours. The day of the experiment the mediawas removed and attaching cells were treated with trypsin for 2 minutes,cells were then collected, washed with PBS (phosphate buffer saline),spin down, resuspended in 2% heat inactivated FBS with RPMI at aconcentration of 1×10⁶ cells/ml, and 100 μl was plated in a 96 wellplate. Compounds were dissolved in dimethyl sulfoxide (DMSO) and addedto the culture medium to achieve desired concentration (e.g. 100, 30,10, 3, 1, 0.3 or 0.1 μM). Cells were incubated with compounds for 4hours. Cell free supernatant was collected and the production of IL-1βwas evaluated by ELISA. A vehicle only control was run concurrently witheach experiment. Final DMSO concentration was 1%. Compounds exhibit adose-related increase of IL-1β production in PMA-differentiated THP-1cells.

Measurement of IL-1β Production in PMA-Differentiated THP-1 Cells(Alternative Procedure)

THP-1 cells were purchased from the American Type Culture Collection andsub-cultured according to instructions from the supplier. Prior toexperiments, cells were cultured in RPMI 1640 containing 10% heatinactivated FBS, penicillin (100 units/ml), streptomycin (100 μg/ml),HEPES (10 mM) and sodium pyruvate (1 mM) and maintained in log phaseprior to experimental setup. Prior to the experiment, THP-1 cells weretreated with PMA (Phorbol 12-myristate 13-acetate) (20 μg/ml) overnight.The day of the experiment, the media was removed and attached cells weretreated with trypsin for 2 minutes, cells were then collected, washedwith PBS (phosphate buffer saline), pelleted by centrifugation andresuspended in 2% heat inactivated FBS with RPMI at a concentration of50,000 cells/well in a 384 well plate. Cell free supernatant wascollected and the production of IL-1β was evaluated by ELISA. Compoundswere dissolved in dimethyl sulfoxide (DMSO) and added to the culturemedium to achieve desired concentration (e.g. 100, 30, 10, 3, 1, 0.3 or0.1 μM). Cells were incubated with compounds for 2 hours. A vehicle onlycontrol was run concurrently with each experiment. Final DMSOconcentration was 1%. Compounds exhibit a dose-related increase of IL-1βproduction in PMA-differentiated THP-1 cells.

Measurement of IL-1β Production—hTRF Protocol (Second AlternativeProcedure)

Serial dilutions of compounds in DMSO were added to low volume 384 wellplates at 100 nl/well using an ECHO 550 acoustic dispenser (Labcyte) toachieve final starting concentration of 10 μM in assay.

THP-1 cells in RPMI (Gibco, 11875) media with 10% FBS at a density of1×10⁶ cell/ml in a T175 flask were treated with a final concentration ofphorbol 12-myristate 13-acetate (PMA) (Sigma,P1585) of 50 ng/mlovernight at 37° C. at 5% CO₂ for differentiation. Cells were harvestedthe next day after rinsing well with dPBS using 0.5% trypsin. A cellsolution was prepared of 1×10⁶ cells/ml for 50,000 cells in 50 μl/wellin RPMI media with 2% FBS. Cells were plated using a multichannelpipette onto the compound dilutions in Greiner, 384 well, black clearbottom tissue culture treated plates (781090). The plates were incubatedin 37° C. incubator at 5% CO₂ for 2 hours.

After the 2 hour incubation, the cell plates were spun in the centrifugefor 5 minutes at 1200 rpm. Using the Felix (CyBio), 8 μl of thesupernatant was transferred to 384 well, low volume, white proxy plates.(Perkin Elmer, 6008230). A human IL1beta hTRF kit was used to analyzethe supernatant (CISBIO, 62HIL1BPEG). The kit instructions were followedfor preparing the IL1Beta standard curve and then the antibodies fromthe kit were diluted 1:40 rather than 1:20 as kit instructed. Oncecombined, the antibodies were added across the plates, 5 μl/well. Theplates were sealed and incubated at 4° C. overnight. The plates werethen read on the Perkin Elmer EnVision at 665/615 nm using the hTRFlaser. Compounds exhibited a dose-related increase of IL-1β production.

Measurement of IL-1β Production—Human Whole Blood Assay

Serial dilutions of compounds in DMSO were added to low volume 384 wellplates at 100 nl/well using an ECHO 550 acoustic dispenser (Labcyte) toachieve final starting concentration of 10 μM in assay.

Human venous whole blood obtained from healthy donors was pre-treatedwith LPS (Invivogen, Cat #tlrl-eblps) at 1 ng/ml for four hours at 37°C. in a humidified 95% air/5% CO₂ incubator. Primed blood was added tothe compound plate and incubated for additional 4 hours at 37° C.IL-1beta in the supernatants was measured using AlphLISA kit (Cat#AL220) according to manufacturer's instructions. Compounds exhibited adose-related increase of IL-1β production. EC50 was determined usingprimed but untreated blood as baseline.

Measurement of IL-1β Production—mouse hTRF Protocol

Immortalized mouse macrophages derived from C57BL/6 mice were obtainedfrom Ericke Latz, University of Bonn/University of MassachusettsWorchester, MA. The cells were harvested using 0.05% Trypsin and washedwith PBS. Cell were plated at 30,000 cells per well in 25 ul in DMEM(Gibco, 11965) supplemented with 2% FBS and incubated for 10 minutes at37° C. at 5% CO₂. LPS-EB (Invivogen, tlr-eblps) was added to a finalconcentration of 200 ng/ml at 5 ul/well and cells were incubated for 2hours at 37° C. at 5% CO₂.

Serial dilutions of compounds in DMSO were added to cells in low volume384 well plates at 60 nl/well using an ECHO 550 acoustic dispenser(Labcyte) to achieve final starting concentration of 50 uM in assay andincubated with compounds for additional 2 hours at 37° C. at 5% CO₂.

After the 2 hour incubation, the cell plates were spun in the centrifugefor 5 minutes at 1200 rpm. Using the Felix (CyBio), 8 ul of thesupernatant was transferred to 384 well, low volume, white proxy plates.(Perkin Elmer, 6008230). A human IL1beta hTRF kit was used to analyzethe supernatant (CISBIO, 62MIL1BPEH). The kit instructions were followedfor preparing the IL1Beta standard curve (the antibodies from the kitwere diluted 1:40 rather than 1:20 as kit instructed). Once combined,the antibodies were added across the plates at 5 ul/well. The plateswere sealed and incubated at 4° C. overnight. The plates were read onthe Perkin Elmer EnVision at 665/615 nm using the hTRF laser. Data wasthen converted to pg/ml of Il1Beta. Compounds exhibited a dose-relatedincrease of IL-1β production.

In Vitro Human TLR7 and TLR8 Binding Reporter Assays

Logarithmically-growing human HEK-Blue cells co-expressing a TLR7 orTLR8 gene and a NF-kB/AP1-inducible SEAP (secreted embryonic alkalinephosphatase; Invivogen, San Diego, CA) reporter gene are added toindividual wells of a 384-well plate (15,000 cells per 20 μL per well)and maintained for 24 h at 37° C., 5% CO₂. Test compounds or DMSO aredistributed to separate wells the next day using acoustic liquidhandling technology (100 nL per well) and cells are subsequentlyincubated for 18 h at 37° C., 5% CO₂. Cellular SEAP production ismeasured using an Envision plate reader instrument thirty minutes afteradding freshly-made Quanti-Blue reagent (prepared by followingmanufacturer instructions; Invivogen, San Diego, CA) to the HEK-Blue TLRNf-kB-SEAP cell reactions. All EC₅₀ values (half-maximal effectiveconcentration) are determined using proprietary data analysis software.Normalized EC₅₀ value=absolute value determined by setting 100% Ymaxusing a reference standard RLU (relative light unit) values from cellstreated with 50 μM of the reference standard.

EXAMPLES

To further illustrate the foregoing, the following non-limiting,exemplary synthetic schemes are included. Variations of these exampleswithin the scope of the claims are within the purview of one skilled inthe art and are considered to fall within the scope of the invention asdescribed, and claimed herein. The reader will recognize that theskilled artisan, provided with the present disclosure, and skill in theart is able to prepare and use the invention without exhaustiveexamples.

HPLC/MS and Preparatory/Analytical HPLC Methods Employed inCharacterization or Purification of Examples

Analytical HPLC/MS was performed using the following methods:

Method A: Column: Waters XBridge C18, 2.1 mm×50 mm, 1.7 m particles;Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid;Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid;Temperature: 50° C.; Gradient: 0% B to 100% B over 3 min, then a 0.50min hold at 100% B; Flow: 1 mL/min; Detection: MS and UV (220 nm).

Method B: Column: Waters XBridge C18, 2.1 mm×50 mm, 1.7 m particles;Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammonium acetate;Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammonium acetate;Temperature: 50° C.; Gradient: 0% B to 100% B over 3 min, then a 0.50min hold at 100% B; Flow: 1 mL/min; Detection: MS and UV (220 nm).

Method C: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm, 1.7-μmparticles; Mobile Phase A: water with 0.05% TFA; Mobile Phase B:acetonitrile with 0.05% TFA; Temperature: 50° C.; Gradient: 2-98% B over1.0 minute, then a 0.50-minute hold at 98% B; Flow: 0.80 mL/min;Detection: UV at 254 nm.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Chemical shifts are reported in parts per million (ppm) downfield frominternal tetramethylsilane (TMS) or from the position of TMS inferred bythe deuterated NMR solvent. Apparent multiplicities are reported as:singlet-s, doublet-d, triplet-t, quartet-q, or multiplet-m. Peaks whichexhibit broadening are further denoted as br. Integrations areapproximate. It should be noted that integration intensities, peakshapes, chemical shifts and coupling constants can be dependent onsolvent, concentration, temperature, pH, and other factors. Further,peaks which overlap with or exchange with water or solvent peaks in theNMR spectrum may not provide reliable integration intensities. In somecases, NMR spectra are obtained using water peak suppression, which mayresult in overlapping peaks not being visible or having altered shapeand/or integration.

Example 1. tert-Butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

1A. 7-Bromo-3-nitroquinolin-4-ol

To a homogeneous solution of 7-bromoquinolin-4-ol (2.00 g, 8.93 mmol) inpropionic acid (15.36 ml, 205 mmol) stirring at 120° C. was added nitricacid (750 uL) at a rate of 50 uL/5 min. After the addition was complete,the heterogeneous reaction mixture was stirred at 120° C. for 2 h. Aftercooling to room temperature, ethanol (30 mL, 200 proof) was added, andthe suspension was stirred for 20 min. The solid was collected by vacuumfiltration, washed with ethanol (3×15 mL), and dried under reducedpressure to give 7-bromo-3-nitroquinolin-4-ol (1.87 g, 6.95 mmol, 78%yield) as a tan solid.

LC/MS [M+H]⁺=269.1 and 271.1.

1B. 7-Bromo-4-chloro-3-nitroquinoline

A heterogeneous mixture of 7-bromo-3-nitroquinolin-4-ol (4.00 g, 14.87mmol), POCl₃ (21 mL, 223 mmol), and N,N-dimethylformamide (1.0 mL) undernitrogen was immersed in an oil bath heated to 70° C. The resultinghomogeneous reaction mixture was stirred at 70° C. for 4 h. Theresulting precipitate wa collected by vacuum filtration. The collectionflask was changed, and the solid was washed with water. The solid wasdissolved in dichloromethane, washed with a saturated aqueous solutionof sodium carbonate, and dried over anhydrous sodium sulfate. The waterwashes were extracted with dichloromethane (3×), and the combinedorganic layers were was with a saturated aqueous solution of sodiumcarbonate, and dried over anhydrous sodium sulfate. The combined organiclayers were concentration under reduce pressure to afford7-bromo-4-chloro-3-nitroquinoline (3.06 g, 10.64 mmol, 72% yield) as anoff-white solid. LC/MS [M+H]⁺=287.0 and 289.0.

1C. 7-Bromo-3-nitroquinolin-4-amine

A solution of 7-bromo-4-chloro-3-nitroquinoline (1.50 g, 5.22 mmol) andammonium hydroxide (2.18 mL, 15.7 mmol) in acetonitrile (15 mL) in asealed tube was immersed in an oil bath at 40° C. and stirred for 3 h,during which time the reaction mixture solidified. The mixture wasdiluted with methanol and dispersed with sonication. The solid wascollected by vacuum filtration and washed with methanol. The filtratewas concentrated and combined with the solid to give7-bromo-3-nitroquinolin-4-amine (1.40 g, 5.17 mmol, 99% yield) as ayellow solid. LC/MS [M+H]⁺=268.0 and 270.0.

1D. 7-Bromoquinoline-3,4-diamine

A heterogeneous mixture of 7-bromo-3-nitroquinolin-4-amine (1.40 g, 5.22mmol) and tin(II) chloride dihydrate (4.24 g, 18.8 mmol) in ethylacetate (30 mL) was heated at reflux for 3 h. The reaction remainedheterogeneous, but was complete when analyzed. The reaction mixture wasdiluted with ethyl acetate, washed with 2.5 N aqueous sodium hydroxideuntil the aqueous layer was clear (3×), and washed with brine. Theorganic layer was collected, and the aqueous layers were sequentiallyextracted with ethyl acetate (3×). The combine organic layer was driedover anhydrous sodium sulfate and concentrated under reduced pressure togive 7-bromoquinoline-3,4-diamine (1.21 g, 5.08 mmol, 97% yield) as ayellow solid. LC/MS [M+H]⁺=238.1 and 240.1.

1E. tert-Butyl(S)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (0.665 g, 2.79 mmol) and(S)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (0.813 g, 3.55mmol) in ethyl acetate (15 mL) at room temperature was added pyridine(1.13 mL, 14.0 mmol) followed by 1-propanephosphonic anhydride (50 wt %in DMF) (3.33 mL, 5.59 mmol). During the addition, a gum formed, butovertime it dissolved. The mixture was diluted with ethyl acetate,washed with 1 N aqueous sodium hydroxide (2×), washed with 10% lithiumchloride (2×), and washed with brine. The organic layer was collected,and the aqueous layers were sequentially extracted with ethyl acetate(3×). The combined organic layers were dried over anhydrous sodiumsulfate and concentrated under reduced pressure to give tert-butyl(S)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(1.26 g, 2.80 mmol, 100% yield) as a tan solid. LC/MS [M+H]⁺=449.2 and451.2.

1F. tert-Butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl(S)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(1.26 g, 2.80 mmol) and sodium hydroxide (1.12 g, 28.0 mmol) in ethanol(20 mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2 h. The reaction mixture was concentrated under reducedpressure, and residue was dissolved in ethyl acetate and water. Theorganic layer collected, washed with water, and washed with brine. Theaqueous layers were sequentially extracted with ethyl acetate (3×), andthe combined organic layer were dried over anhydrous sodium sulfate.Concentration under reduced pressure afforded tert-butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(1.21 g, 2.81 mmol, 100% yield) as a yellow solid. LC/MS [M+H]⁺=431.2and 433.2.

1G. tert-Butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(1.21 g, 2.81 mmol) and mCPBA (1.29 g, 5.61 mmol) in dichloromethane (15mL) was stirred at room temperature for 3 h. To the reaction mixture wasadded ammonium hydroxide (5.0 mL) dropwise followed by tosyl-Cl (1.07 g,5.61 mmol). The resulting reaction mixture was stirred for 60 min. Thehomogeneous reaction mixture was extracted with dichloromethane, and theorganic layer was collected. The small ammonium hydroxide layer wasextracted with dichloromethane (2×). The aqueous layer was diluted withwater, the pH was adjusted to ˜5 with 6N aqueous hydrochloric acid, andextracted with ethyl acetate (3×). The combined organic layers weredried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by ISCO flash silica gelchromatography (40 g column; 0%-10% methanol in dichloromethane) toafford tert-butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.728 g, 1.60 mmol, 57% yield) as a tan solid. LC/MS [M+H]⁺=446.2 and448.2.

Example 1

A mixture of tert-butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.728 g, 1.63 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.633 g,3.26 mmol), and tripotassium phospate (2M in water) (2.45 mL, 4.89 mmol)in dioxane (10 mL) was degassed (3×; vacuum/nitrogen). To the mixturewas added 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride(0.133 g, 0.163 mmol), and the mixture was degassed (3×;vacuum/nitrogen). The reaction was immersed in an oil bath at 85° C. andstirred overnight. The reaction mixture was diluted with ethyl acetateand washed with water and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by ISCO flash chromatography (40 g; 1-10% methanol indichloromethane) to give tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.517 g, 1.18 mmol, 72% yield) as a tan solid.

Example 2.(S)-2-(Piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.517 g, 1.193 mmol, Example 1) and TFA (3 mL) was stirred at roomtemperature for 45 min. The TFA was removed under reduced pressure, andthe residue was azeotroped with dioxane (3×) to give(S)-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.395 g, 1.19 mmol, 99% yield) was a light tan solid.

Example 3.(S)-1-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carbonyl)cyclopropane-1-carbonitrile

To a mixture of(S)-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.020 g, 0.060 mmol, Example 2), 1-cyanocyclopropane-1-carboxylic acid(10.0 mg, 0.090 mmol), and Hunig's Base (0.105 mL, 0.600 mmol) inN,N-dimethylformamide (1.9 mL) was added 1-propanephosphonic anhydride(50 wt % in DMF) (0.039 mL, 0.066 mmol). The reaction was placed on ashaker plate for 60 min. The reaction mixture was filtered through aWhatman 0.45 um PTFE w/GMF filter disc and submitted to SCP forpurification. The crude material was purified via preparative LC/MS withthe following conditions: Column: XBridge C18, 200 mm×19 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile: water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile: water with 0.1%trifluoroacetic acid; Gradient: a 0-minute hold at 0% B, 0-40% B over 20minutes, then a 4-minute hold at 100% B; Flow Rate: 20 mL/min; ColumnTemperature: 25 C. Fraction collection was triggered by MS signals.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give(S)-1-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carbonyl)cyclopropane-1-carbonitrile(9 mg, 30% yield) as a yellow solid.

Alternative Preparation of Example 3

To a mixture of(S)-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.076 g, 0.228 mmol, Example 2), 1-cyanocyclopropane-1-carboxylic acid(0.038 g, 0.342 mmol), and Hunig's Base (0.398 mL, 2.28 mmol) inN,N-dimethylformamide (3 mL) was added 1-propanephosphonic anhydride (50wt % in DMF) (0.149 mL, 0.251 mmol). The reaction was stirred at roomtemperature overnight. The reaction mixture was disluted with ethylacetate, washed with water, washed with 1N aqueous sodium hydroxide, andwashed brine. The organic layer was collected, and the aqueous layerswere sequentially extracted with ethyl acetate. The combined organiclayers were dried over anhydrous sodium sulfate and concentrated.Purification by ISCO flash silica chromatography (4 g column; 0-10%methanol in dichloromethane) afforded(S)-1-(3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carbonyl)cyclopropane-1-carbonitrile(0.056 g, 0.126 mmol, 55.3% yield) as a yellow solid.

Example 4.(S)-1-(3-(4-Amino-1-ethyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)propan-1-one

4A. 7-Bromo-N-ethyl-3-nitroquinolin-4-amine

A solution of 7-bromo-4-chloro-3-nitroquinoline (1.50 g, 5.22 mmol, 1B)and ethanamine (2.0 M in MeOH) (5.22 mL, 10.4 mmol) in acetonitrile (16mL) in a sealed tube was immersed in an oil bath at 40° C. and stirredfor 3 h, during which time the reaction mixture solidified. The mixturewas diluted with methanol and dispersed with sonication. The solid wascollected by vacuum filtration and washed with methanol. The filtratewas concentrated and combined with the solid to give7-bromo-N-ethyl-3-nitroquinolin-4-amine (1.55 g, 5.23 mmol, 100% yield)as a yellow solid. LC/MS [M+H]⁺=296.1 and 298.1.

4B. 7-Bromo-N4-ethylquinoline-3,4-diamine

A heterogeneous mixture of 7-bromo-N-ethyl-3-nitroquinolin-4-amine (1.55g, 5.23 mmol) and tin(II) chloride dihydrate (4.25 g, 18.8 mmol) inethyl acetate (30 mL) was heated at reflux for 3 h. The reactionremained heterogeneous, but was complete by analysis. The reactionmixture was diluted with ethyl acetate, washed with 2.5 N aqueous sodiumhydroxide until the aqueous layer was clear (3×), and washed with brine.The organic layer was collected, and the aqueous layers weresequentially extracted with ethyl acetate (3×). The combine organiclayer was dried over anhydrous sodium sulfate and concentrated underreduced pressure to give 7-bromo-N4-ethylquinoline-3,4-diamine (1.27 g,4.77 mmol, 91% yield) as a yellow solid. LC/MS [M+H]⁺=266.1 and 268.1.

4C. tert-Butyl(S)-3-((7-bromo-4-(ethylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate

To a suspension of 7-bromo-N4-ethylquinoline-3,4-diamine (1.27 g, 4.77mmol) and (S)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (1.31g, 5.73 mmol) in ethyl acetate (25 mL) at room temperature was addedpyridine (1.93 mL, 23.9 mmol) followed by 1-propanephosphonic anhydride(50 wt % in DMF) (5.68 mL, 9.54 mmol). During the addition, a gumformed, but overtime it dissolved. The mixture was diluted with ethylacetate, washed with 1 N aqueous sodium hydroxide (2×), washed with 10%lithium chloride (2×), and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydroussodium sulfate and concentrated under reduced pressure to givetert-butyl(S)-3-((7-bromo-4-(ethylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate(2.27 g, 4.75 mmol, 100% yield) as a tan solid. LC/MS [M+H]⁺=477.3 and479.3.

4D. tert-Butyl(S)-3-(7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl(S)-3-((7-bromo-4-(ethylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate(2.27 g, 4.75 mmol) and sodium hydroxide (1.90 g, 47.5 mmol) in EtOH (35mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2 h. The reaction mixture was concentrated under reducedpressure, and the residue was dissolved in ethyl acetate and water. Theorganic layer collected, washed with water, and washed with brine. Theaqueous layers were sequentially extracted with ethyl acetate (3×), andthe combined organic layer were dried over anhydrous sodium sulfate.Concentration under reduced pressure followed by purification by ISCOflash silica gel chromatography (80 g column; 0-10% methanol indichloromethane) afforded tert-butyl(S)-3-(7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(1.88 g, 4.09 mmol, 86% yield) as a light brown solid. LC/MS[M+H]⁺=459.3 and 461.3.

4E. tert-Butyl(S)-3-(4-amino-7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl(S)-3-(7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(1.88 g, 4.09 mmol) and mCPBA (1.88 g, 8.18 mmol) in dichloromethane (22mL) was stirred at room temperature for 3 h. To the reaction mixture wasadded ammonium hydroxide (7.33 mL) dropwise followed by tosyl-Cl (1.56g, 8.18 mmol). The resulting reaction mixture was stirred for 60 min.The homogeneous reaction mixture was extracted with dichloromethane, andthe organic layer was collected. The ammonium hydroxide layer wasdiluted with water and extracted with dichloromethane (3×).Concentration under reduced pressure followed by purification ISCO flashsilica gel chromatography (80 g column; 0%-10% methanol indichloromethane) failed to separate the product from unpurities.Repurification (0-5%) afforded tert-butyl(S)-3-(4-amino-7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.412 g, 0.868 mmol, 21% yield) as a tan solid. LC/MS [M+H]⁺=474.3 and476.3.

4F. tert-Butyl(S)-3-(4-amino-1-ethyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl(S)-3-(4-amino-7-bromo-1-ethyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.406 g, 0.856 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.332 g,1.71 mmol), and tripotassium phosphate (2M in water) (1.3 mL, 2.57 mmol)in dioxane (6 mL) was degassed (3×; vacuum/nitrogen). To the mixture wasadded 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride(0.070 g, 0.086 mmol), and the mixture degassed (3×; vacuum/nitrogen).The reaction was immersed in an oil bath at 85° C. and stirredovernight. The reaction mixture was diluted with ethyl acetate andwashed with water and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by ISCO flash chromatography (40 g; 1-10% methanol indichloromethane) to give tert-butyl(S)-3-(4-amino-1-ethyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.327 g, 0.701 mmol, 82% yield) as a tan solid. LC/MS [M+H]⁺==462.5.

4G.(S)-1-Ethyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(S)-3-(4-amino-1-ethyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.327 g, 0.708 mmol) and TFA (3 mL) was stirred at room temperature for45 min. The TFA was removed under reduced pressure, and the residue wasazeotroped with dioxane (3×) to give(S)-1-ethyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.256 g, 0.708 mmol, 100% yield) as a light tan solid.

Example 4

To a mixture of(S)-1-ethyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.021 g, 0.058 mmol), propionic acid (6.51 μl, 0.087 mmol), and Hunig'sBase (0.101 mL, 0.581 mmol) in N,N-dimethylformamide (1.9 mL) was added1-propanephosphonic anhydride (50 wt % in DMF) (0.038 mL, 0.064 mmol).The reaction was placed on a shaker plate for 60 min. The reactionmixture was filtered through a Whatman 0.45 um PTFE w/GMF filter discand submitted to SCP for purification. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammonium acetate; Gradient: a 0-minute hold at 8% B,8-48% B over 23 minutes, then a 6-minute hold at 100% B; Flow Rate: 20mL/min; Column Temperature: 25 C. Fraction collection was triggered byMS signals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give(S)-1-(3-(4-amino-1-ethyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)propan-1-one(22 mg, 87% yield).

Example 5.(S)-1-(3-(4-Amino-1-isopropyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)propan-1-one

5A. 7-Bromo-N-isopropyl-3-nitroquinolin-4-amine

A solution of 7-bromo-4-chloro-3-nitroquinoline (1.00 g, 3.48 mmol, 1B)and isopropylamine (0.596 mL, 6.96 mmol) in acetonitrile (10 mL) in asealed tube was immersed in an oil bath at 40° C. and stirred for 3 h,during which time the reaction mixture solidified. The mixture wasdiluted with methanol and dispersed with sonication. The solid wascollected by vacuum filtration and washed with methanol. The filtratewas concentrated and combined with the solid to give7-bromo-N-isopropyl-3-nitroquinolin-4-amine (1.08 g, 3.48 mmol, 100%yield) as a yellow solid. LC/MS [M+H]⁺=310.2 and 312.2.

5B. 7-Bromo-N4-isopropylquinoline-3,4-diamine

A heterogeneous mixture of 7-bromo-N-isopropyl-3-nitroquinolin-4-amine(1.08 g, 3.48 mmol) and tin(II) chloride dihydrate (2.83 g, 12.5 mmol)in ethyl acetate (20 mL) was heated at reflux for 3 h. The reactionremained heterogeneous, but was complete by analysis. The reactionmixture was diluted with ethyl acetate, washed with 2.5 N aqueous sodiumhydroxide until the aqueous layer was clear (3×), washed with water, andwashed with brine. The organic layer was collected, and the aqueouslayers were sequentially extracted with ethyl acetate (3×). The combinedorganic layers were dried over anhydrous sodium sulfate and concentratedunder reduced pressure to give 7-bromo-N4-isopropylquinoline-3,4-diamine(0.831 g, 2.97 mmol, 85% yield) as a yellow solid. LC/MS [M+H]⁺=280.2and 282.2.

5C. tert-Butyl(S)-3-((7-bromo-4-(isopropylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate

To a suspension of 7-bromo-N4-isopropylquinoline-3,4-diamine (0.831 g,2.97 mmol) and (S)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid(0.816 g, 3.56 mmol) in ethyl acetate (16 mL) at room temperature wasadded pyridine (1.199 mL, 14.83 mmol) followed by 1-propanephosphonicanhydride (50 wt % in DMF) (3.53 mL, 5.93 mmol). During the addition, agum formed, but overtime it dissolved. The mixture was diluted withethyl acetate, washed with 1 N aqueous sodium hydroxide (2×), washedwith 10% lithium chloride (2×), and washed with brine. The organic layerwas collected, and the aqueous layers were sequentially extracted withethyl acetate (3×). The combined organic layers were dried overanhydrous sodium sulfate and concentrated under reduced pressure to givetert-butyl(S)-3-((7-bromo-4-(isopropylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate(1.46 g, 2.97 mmol, 100% yield) as a light brown solid. LC/MS[M+H]⁺=491.3 and 493.3.

5D. tert-Butyl(S)-3-(7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl(S)-3-((7-bromo-4-(isopropylamino)quinolin-3-yl)carbamoyl)piperidine-1-carboxylate(0.779 g, 1.585 mmol) and sodium hydroxide (0.634 g, 15.85 mmol) inethanol (15 mL) was immersed in an oil bath at 80° C. and heatedovernight. The reaction mixture was concentrated under reduced pressure,and the residue was dissolved in ethyl acetate and water. The organiclayer collected, washed with water, and washed with brine. The aqueouslayers were sequentially extracted with ethyl acetate (3×), and thecombined organic layer were dried over anhydrous sodium sulfate.Concentration under reduced pressure followed by purification by ISCOflash silica gel chromatography (80 g column; 0-10% methanol indichloromethane) afforded tert-butyl(S)-3-(7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.430 g, 0.908 mmol, 57% yield) as a light brown solid. LC/MS[M+H]⁺=473.3 and 475.3.

5E. tert-Butyl(S)-3-(4-amino-7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl(S)-3-(7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.430 g, 0.908 mmol) and mCPBA (0.418 g, 1.817 mmol) in dichloromethane(5 mL) was stirred at room temperature for 3 h. To the reaction mixturewas added ammonium hydroxide (1.67 mL) dropwise followed by tosyl-Cl(0.346 g, 1.82 mmol). The resulting reaction mixture was stirred for 60min. The homogeneous reaction mixture was extracted withdichloromethane, and the organic layer was collected. The ammoniumhydroxide layer was diluted with water and extracted withdichloromethane (3×). Concentration under reduced pressure followed bypurification by ISCO flash silica gel chromatography (40 g column; 0%-5%methanol in dichloromethane) afforded tert-butyl(S)-3-(4-amino-7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.188 g, 0.385 mmol, 42% yield) as a tan solid. LC/MS [M+H]⁺=488.3 and490.3

5F. tert-Butyl(S)-3-(4-amino-1-isopropyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl(S)-3-(4-amino-7-bromo-1-isopropyl-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.305 g, 0.624 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.242 g,1.25 mmol), and tripotassium phospate (2M in water) (0.937 mL, 1.87mmol) in dioxane (4 mL) was degassed (3×; vacuum/nitrogen). To themixture was added 1,1′-Bis(di-tert-butylphosphino)ferrocene palladiumdichloride (0.051 g, 0.062 mmol), and the mixture degassed (3×;vacuum/nitrogen). The reaction was immersed in an oil bath at 85° C. andstirred overnight. The reaction mixture was diluted with ethyl acetate,washed with water, and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydroussodium sulfate and concentrated under reduced pressure. The residue waspurified by ISCO flash chromatography (40 g; 1-10% methanol indichloromethane) to give tert-butyl(S)-3-(4-amino-1-isopropyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.236 g, 0.496 mmol, 79% yield) as a tan solid. LC/MS [M+H]⁺=476.5.

5G.(S)-1-Isopropyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(S)-3-(4-amino-1-isopropyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.237 g, 0.498 mmol) and TFA (3 mL) was stirred at room temperature for45 min. The TFA was removed under reduced pressure, and the residue wasazeotroped with dioxane (3×) to give(S)-1-isopropyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.187 g, 0.488 mmol, 98% yield) was a light tan solid. LC/MS[M+H]⁺=376.4.

Example 5

To a mixture of(S)-1-isopropyl-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.021 g, 0.056 mmol), butyric acid (7.72 μl, 0.084 mmol), and Hunig'sBase (0.098 mL, 0.559 mmol) in N,N-dimethylformamide (1.9 mL) was added1-propanephosphonic anhydride (50 wt % in DMF) (0.037 mL, 0.062 mmol).The reaction was placed on a shaker plate for 60 min. The reactionmixture was filtered through a Whatman 0.45 um PTFE w/GMF filter discand purified via preparative LC/MS with the following conditions:Column: XBridge C18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile: water with 10-mM ammonium acetate; Gradient: a 0-minutehold at 12% B, 12-40% B over 35 minutes, then a 4-minute hold at 100% B;Flow Rate: 20 mL/min; Column Temperature: 25 C. Fraction collection wastriggered by MS signals. Fractions containing the desired product werecombined and dried via centrifugal evaporation to giveS)-1-(3-(4-amino-1-isopropyl-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)butan-1-one(10 mg, 39% yield).

Example 6. tert-Butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

6A. tert-Butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (1.0 g, 4.20 mmol) and(R)-1-(tert-butoxycarbonyl)piperidine-3-carboxylic acid (1.223 g, 5.33mmol) in ethyl acetate (21 mL) at room temperature was added pyridine(1.699 mL, 21.00 mmol) followed by dropwise addition of1-propanephosphonic anhydride (50 wt % in DMF) (5.00 mL, 8.40 mmol).During the addition, a small amount of gum formed but overtime itdissolved. The mixture was diluted with ethyl acetate, washed with 1 Naqueous sodium hydroxide (2×), washed with 10% lithium chloride (2×),and washed with brine. The organic layer was collected, and the aqueouslayers were sequentially extracted with ethyl acetate (3×). The combinedorganic layers were dried over anhydrous magnesium sulfate andconcentrated under reduced pressure to give tert-butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(1.85 g, 4.12 mmol, 98% yield) as a light beige solid. LC/MS[M+H]⁺=449.2 and 450.9. ¹H-NMR (400 MHz, CDCl₃) δ 8.51-8.46 (m, 1H),8.15 (d, J=1.8 Hz, 1H), 7.68-7.62 (m, 1H), 7.55 (dd, J=8.9, 1.8 Hz, 1H),5.20 (br s, 2H), 3.87-3.70 (m, 1H), 3.57-3.40 (m, 1H), 2.76-2.65 (m,1H), 2.29-2.12 (m, 1H), 2.00-1.84 (m, 1H), 1.82-1.67 (m, 1H), 1.61-1.53(m, 1H), 1.48 (s, 9H).

6B. tert-Butyl(R)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(2.61 g, 5.81 mmol) and sodium hydroxide (2.323 g, 58.1 mmol) in EtOH(41.5 ml) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2 h under a condensor of nitrogen and was concentrated underreduced pressure. The residue was dissolved in ethyl acetate and water.The organic layer was collected and washed with water and brine. Theaqueous layers were sequentially extracted with ethyl acetate (3×), andthe combined organic layer was dried over anhydrous magnesium sulfate.Concentration under reduced pressure afforded tert-butyl(R)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(2.39 g, 5.54 mmol, 95% yield) as a gold solid. LC/MS [M+H]⁺=431.3 and433.3.

6C. tert-Butyl(R)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl(R)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(1.21 g, 2.81 mmol) and mCPBA (1.291 g, 5.61 mmol) in dichloromethane(15 mL) was stirred at room temperature for 3 h. To the reaction mixturewas added ammonium hydroxide (5.00 mL) dropwise followed by tosyl-Cl(1.07 g, 5.61 mmol). The resulting reaction mixture was stirred for 60min. The homogeneous reaction mixture was extracted with dichloromethaneand the organic layer was collected. The small ammonium hydroxide layerwas extracted with dichloromethane (2×). The aqueous layer was dilutedwith water, the pH was adjusted to ˜5 with 6N aqueous hydrochloric acidand was extracted with ethyl acetate (3×). The combined organic layerswere dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The residue was purified by ISCO flash silica gelchromatography (40 g column; 0%-10% methanol in dichloromethane) toafford tert-butyl(R)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.70 g, 1.60 mmol, 56% yield) as a tan solid. LC/MS [M+H]⁺=446.4 and448.4.

Example 6

A mixture of tert-butyl(R)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.67 g, 1.50 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.583 g,3.00 mmol), and tripotassium phospate (2M in water) (2.25 mL, 4.50 mmol)in dioxane (9.2 mL) was degassed (3×; vacuum/nitrogen). To the mixturewas added 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride(0.123 g, 0.150 mmol), and the mixture was degassed (3×;vacuum/nitrogen). The reaction was immersed in an oil bath at 80° C. andstirred overnight. The reaction mixture was diluted with ethyl acetateand washed with water and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydrousmagnesium sulfate and concentrated under reduced pressure. The residuewas purified by ISCO flash chromatography (40 g; 0-20% methanol indichloromethane) and a small sample was purified by preparative LC/MSwith the following conditions. Column: XBridge C18, 200 mm×19 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10-mM ammoniumacetate; Gradient: a 0-minute hold at 13% B, 13-53% B over 20 minutes,then a 4-minute hold at 100% B; Flow Rate: 20 mL/min; ColumnTemperature: 25° C. Fraction collection was triggered by MS and UVsignals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give tert-butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.400 g, 0.92 mmol, 62% yield) as a tan solid.

Example 7.(R)-2-(Piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.40 g, 0.923 mmol) and TFA (2.31 mL) was stirred at room temperaturefor 60 min. The TFA was removed under reduced pressure, and the residuewas azeotroped with dioxane (3×). The crude material was purified viapreparative LC/MS with the following conditions: Column: XBridge C18,200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: a 2-minute hold at 0% B, 0-40%B over 20 minutes, then a 4-minute hold at 100% B; Flow Rate: 20 mL/min;Column Temperature: 25° C. Fraction collection was triggered by MSsignals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give(R)-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.31 g, 0.71 mmol, 98% yield) as a light tan solid.

Example 8.(R)-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)(cyclobutyl)methanone

To a mixture of(R)-2-(piperidin-3-yl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.020 g, 0.060 mmol), cyclobutanecarboxylic acid (8.60 μl, 0.090 mmol),and Hunig's base (0.105 mL, 0.600 mmol) in N,N-dimethylformamide (1.9mL) was added 1-propanephosphonic anhydride (50 wt % in DMF) (0.039 mL,0.066 mmol). The reaction was placed on a shaker plate for 60 min. Thereaction mixture was filtered through a Whatman 0.45 um PTFE w/GMFfilter disc. The crude material was purified via preparative LC/MS withthe following conditions. Column: XBridge C18, 200 mm×19 mm, 5-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammoniumacetate; Gradient: a 0-minute hold at 6% B, 6-46% B over 20 minutes,then a 4-minute hold at 100% B; Flow Rate: 20 mL/min; ColumnTemperature: 25° C. Fraction collection was triggered by MS signals.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give(R)-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidin-1-yl)(cyclobutyl)methanone(6 mg, 22% yield).

Example 9.(S)-1-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidin-1-yl)propan-1-one

9A. tert-butyl(S)-3-((4-Amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (0.566 g, 2.38 mmol) and(S)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (0.650 g, 3.02mmol) in ethyl acetate (15 mL) at room temperature was added pyridine(0.962 mL, 11.89 mmol) followed by 1-propanephosphonic anhydride (50 wt% in EtOAc) (2.83 mL, 4.76 mmol). After 60 min, the reaction was stillhomogeneous. Dimethylformamide (1 mL) was added, and the reactionmixture was stirred for 6 min, during which time it became homogeneousThe mixture was diluted with ethyl acetate, washed with 1 N aqueoussodium hydroxide (2×), washed with 10% lithium chloride (2×), and washedwith brine. The organic layer was collected, and the aqueous layers weresequentially extracted with ethyl acetate (3×). The combined organiclayers were dried over anhydrous sodium sulfate and concentrated underreduced pressure to give tert-butyl(S)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate(1.04 g, 2.38 mmol, 100% yield) as a tan solid. LC/MS [M+H]⁺=435.2 and437.2.

9B. tert-Butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

A mixture of tert-butyl(S)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate(1.035 g, 2.378 mmol) and sodium hydroxide (0.951 g, 23.78 mmol) in EtOH(12 mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2 h. After cooling to room temperature, the reaction mixturewas concentrated under reduced pressure, and residue was dissolved inethyl acetate and water. The organic layer collected and washed withbrine. The aqueous layers was sequentially extracted with ethyl acetate(3×), and the combined organic layer were dried over anhydrous sodiumsulfate. Concentration under reduced pressure afforded tert-butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.906 g, 2.17 mmol, 91% yield) as a yellow solid. LC/MS [M+H]⁺=417.2and 419.2.

9C. tert-Butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

A solution of tert-butyl(S)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.906 g, 2.171 mmol) and mCPBA (0.999 g, 4.34 mmol) in dichloromethane(12 mL) was stirred at room temperature for 3 h. To the reaction mixturewas added ammonium hydroxide (4.00 mL) dropwise followed by tosyl-Cl(0.828 g, 4.34 mmol). The resulting reaction mixture was stirred for 60min. The homogeneous reaction mixture was diluted with dichloromethane,and the organic layer was collected. The small ammonium hydroxide layerwas extracted with ethyl acetate (2×). The aqueous layer was dilutedwith water, the pH was adjusted to ˜5 with 6N aqueous hydrochloric acid,and extracted with ethyl acetate (3×). The combined organic layers weredried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by ISCO flash silica gelchromatography (40 g column; 0%-10% methanol in dichloromethane) toafford tert-butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.510 g, 1.17 mmol, 54% yield) as a tan solid. LC/MS [M+H]⁺=432.2 and434.2.

9D. tert-Butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

A mixture of tert-butyl(S)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.417 g, 0.965 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.374 g,1.929 mmol), and ttripotassium phospate (2M in water) (1.5 mL, 2.89mmol) in dioxane (8 mL) was degassed (3×; vacuum/nitrogen). To themixture was added 1,1′-bis(di-tert-butylphosphino)ferrocene palladiumdichloride (0.079 g, 0.096 mmol), and the mixture degassed (3×;vacuum/nitrogen). The reaction was immersed in an oil bath at 95° C. andstirred overnight. The reaction mixture was diluted with ethyl acetateand washed with water. The organic layer was collected, and the aqueouslayer was extracted with ethyl acetate (2×). The aqueous layer hadsignificant precipitated product so the pH was adjusted to ˜5, and themixture was diluted with dichloromethane and filtered under reducedpressure. The solid was washed with water, washed with dichloromethaneand dried well. The solid was suspended in methanol, with sonication,and stirred for overnight. The suspension was filtered under reducedpressure, and the solid was dried to give tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.238 g, 0.562 mmol, 58% yield) as a light tan solid. LC/MS[M+H]⁺=420.3.

The methanol filtrate was concentrated to give tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.029 g, 0.069 mmol, 7.2% yield) as a light tan solid. LC/MS[M+H]⁺=420.4.

The organic layer form the aqueous/dichloromethane filtrate wascollected, and the organic layer was extracted ethyl acetate (3×). Thecombined combined organic layers were washed with brine, and the driedover anhydrous sodium sulfate. Both the dichloromethane layer and theethyl layers were concentrated to give tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.138 g, 0.329 mmol, 34% yield) as a brown solid. LC/MS [M+H]⁺=420.4.

9E.(S)-7-(1H-Pyrazol-3-yl)-2-(pyrrolidin-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

A mixture of tert-butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.238 g, 0.567 mmol) and TFA (3 mL) was stirred at room temperature for45 min. The TFA was removed under reduced pressure, and the residue wasazeotroped with dixoane (3×) to give(S)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.181 g, 0.567 mmol, 100% yield) as a tan solid. LC/MS [M+H]⁺=320.3.

Example 9

To a mixture of(S)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine(0.022 g, 0.069 mmol), propionic acid (6.69 μl, 0.090 mmol), and Hunig'sbase (0.120 mL, 0.689 mmol) in N,N-dimethylformamide (1.8 mL) was added1-propanephosphonic anhydride (50 wt % in DMF) (0.045 mL, 0.076 mmol).The reaction was placed on a shaker plate for 60 min. The reactionmixture was filtered through a Whatman 0.45 um PTFE w/GMF filter discand submitted to SCP for purification. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: XBridgeC18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: a 0-minute hold at 0% B,0-40% B over 20 minutes, then a 4-minute hold at 100% B; Flow Rate: 20mL/min; Column Temperature: 25 C. Fraction collection was triggered byMS signals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give tert-Butyl(S)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(5 mg, 20% yield).

Example 10. tert-Butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

10A. tert-Butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (0.400 g, 1.680 mmol)and (R)-1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (0.470 g,2.184 mmol) in ethyl acetate (6.8 mL) at room temperature was addedpyridine (0.679 mL, 8.40 mmol) followed by 1-propanephosphonic anhydride(50 wt % in EtOAc) (2.00 mL, 3.36 mmol) (During the addition, thereaction mixture became a suspension which redissolved within a coupleof minutes). The homogeneous reaction mixture was stirred at roomtemperature for 30 min. The mixture was diluted with ethyl acetate,washed with 1 N aqueous sodium hydroxide (2×), and washed with brine.The organic layer was collected, and the aqueous layers weresequentially extracted with ethyl acetate (2×). The combined organiclayers were dried over anhydrous sodium sulfate and concentrated underreduced pressure to give tert-butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate(0.45 g, 62% yield) as a tan solid. LC/MS [M+H]⁺=435.0.

10B. tert-Butyl(R)-3-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

To a solution of tert-butyl(R)-3-((4-amino-7-bromoquinolin-3-yl)carbamoyl)pyrrolidine-1-carboxylate(0.45 g, 1.034 mmol) in EtOH (10 mL) was added sodium hydroxide (0.413g, 10.34 mmol). The resulted mixture was heated to 80° C. and keptstirring for 2 h. After cooling to room temperature, the reactionmixture was diluted with dichloromethane and washed with water. Theorganic layer collected, and the aqueous layer was extracted withdichloromethane (2×) and ethyl acetate (2×). The dichloromethane andethyl acetate layers were independently washed with brine and dried overanhydrous sodium sulfate. The combined organic layers were filtered andconcentrated under reduced pressure to give tert-butyl(R)-3-(7-bromo-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylateas a light brown solid. Purification by ISCO (40 g silica gel, 10%MeOH/CH₂Cl₂) to give pure product. LC/MS [M+H]⁺=417.2. ¹H-NMR (400 MHz,HDMSO) δ 9.18-9.14 (m, 1H), 8.34-8.27 (m, 2H), 7.85-7.79 (m, 1H),3.87-3.78 (m, 2H), 3.71-3.61 (m, 1H), 3.56-3.47 (m, 1H), 3.45-3.39 (m,1H), 2.44-2.19 (m, 2H), and 1.47-1.38 (m, 9H).

10C. tert-Butyl(R)-3-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate

A solution of tert-butyl(R)-3-(7-bromo-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(0.5 g, 1.198 mmol) in dichloromethane (15 mL) was treated with mCPBA(0.620 g, 3.59 mmol). The resulted mixture was stirred at roomtemperature for 12 h. To the solution was added ammonium hydroxide(0.233 mL, 5.99 mmol) and tosyl-Cl (0.457 g, 2.396 mmol). The reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas diluted with dichloromethane and washed with water. The organiclayer was collected, and the aqueous layers were sequentially extractedwith dichloromethane (2×). The combined organic layers were dried overanhydrous sodium sulfate and concentrated. Purification by ISCO flashchromatography (24 g column; 0%-10% methanol in dichloromethane)afforded(R)-3-(4-amino-7-bromo-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(400 mg, 77% yield) as a tan solid. LC/MS [M+H]⁺=432.1.

Example 10

Into a 10 mL reaction vial purged and matained with inert atmosphere ofnitrogen, was placed a solution of tert-butyl(R)-3-(4-amino-7-bromo-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(350 mg, 0.810 mmol) in dioxane (10 mL). To a solution was addedpotassium phosphate, tribasic (1.21 mL, 2.43 mmol) (2 M in water),5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (189 mg,0.971 mmol), and bis(tri-tert-butylphosphine)palladium(0) (21 mg, 0.040mmol). The resulting solution was stirred for 12 h at 85° C. Theresulting mixture was concentrated under vacuum, and the residue waspurified by reverse phase, preparative HPLC to yield tert-butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylateas a light yellow solid.

Example 11.(R)-1-(3-(4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)pyrrolidin-1-yl)-2-methylpropan-1-one

A solution of tert-butyl(R)-3-(4-amino-7-(1H-pyrazol-3-yl)-3H-imidazo[4,5-c]quinolin-2-yl)pyrrolidine-1-carboxylate(25 mg, 0.060 mmol) in 1 mL neat TFA was stirred at room temperature for15 min, the reaction was then concentrated and further dried on vacuumpump to give(R)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-3-yl)-3H-imidazo[4,5-c]quinolin-4-amine,LC/MS [M+1]⁺=320.4. To above crude material in THF (2 mL) was addedisobutyric acid (8.28 mg, 0.094 mmol) and triethylamine (0.017 mL, 0.125mmol), and then 1-propanephosphonic anhydride (59.8 mg, 0.094 mmol) (50wt % in EtOAc) was added dropwise. The resulted mixture was stirred atroom temperature for 1 h. Purification by reverse phase, preprative HPLCgave the desired product (5.7 mg, 24% yield).

Example 12. tert-Butyl4-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

12A. tert-Butyl4-((4-Amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (0.323 g, 1.357 mmol)and 1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid (0.395 g, 1.723mmol) in ethyl acetate (12 mL) at room temperature was added pyridine(0.549 mL, 6.78 mmol) followed by 1-propanephosphonic anhydride (50 wt %in DMF) (1.62 mL, 2.71 mmol). During the addition, a gum formed, butovertime it dissolved. The mixture was diluted with ethyl acetate,washed with 1 N aqueous sodium hydroxide (2×), washed with 10% lithiumchloride (2×), and washed with brine. The organic layer was collected,and the aqueous layers were sequentially extracted with ethyl acetate(3×). The combined organic layers were dried over anhydrous sodiumsulfate and concentrated under reduced pressure to give tert-butyl4-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(0.526 g, 1.17 mmol, 86% yield) as a tan solid. LC/MS [M+H]⁺=449.2 and451.2.

12C. tert-Butyl4-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A mixture of tert-butyl4-((4-amino-7-bromoquinolin-3-yl)carbamoyl)piperidine-1-carboxylate(0.526 g, 1.171 mmol) and sodium hydroxide (0.468 g, 11.71 mmol) in EtOH(10 mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2 h and this stirred at room temperature overnight. Thereaction mixture was concentrated under reduced pressure, and residuewas dissolved in ethyl acetate and water. The organic layer collected,washed with water, and washed with brine. The aqueous layers wassequentially extracted with ethyl acetate (3×), and the combined organiclayer were dried over anhydrous sodium sulfate. Concentration underreduced pressure afforded tert-butyl4-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.470 g, 1.09 mmol, 93% yield) as a yellow solid. LC/MS [M+H]⁺=431.2and 433.2.

12D. tert-Butyl4-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate

A solution of tert-butyl4-(7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.470 g, 1.090 mmol) and mCPBA (0.501 g, 2.179 mmol) in dichloromethane(6 mL) was stirred at room temperature for 3 h. To the reaction mixturewas added ammonium hydroxide (2.00 mL) dropwise followed by tosyl-Cl(0.415 g, 2.18 mmol). The resulting reaction mixture was stirred for 60min. The homogeneous reaction mixture was extracted withdichloromethane, and the organic layer was collected. The small ammoniumhydroxide layer was extracted with dichloromethane (2×). The aqueouslayer was diluted with water, the pH was adjusted to ˜5 with 6N aqueoushydrochloric acid, and extracted with ethyl acetate (3×). The combinedorganic layers were dried over anhydrous sodium sulfate and concentratedunder reduced pressure. The residue was purified by ISCO flash silicagel chromatography (12 g column; 0%-10% methanol in dichloromethane) toafford tert-butyl4-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.173 g, 0.388 mmol, 50% yield) as a tan solid. LC/MS [M+H]⁺=446.2 and448.2

Example 12

A mixture of tert-butyl4-(4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.181 g, 0.406 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.157 g,0.811 mmol), and tripotassium phosphate (2M in water) (0.61 mL, 1.22mmol) in dioxane (4 mL) was degassed (3×; vacuum/nitrogen). To themixture was added 1,1′-bis(di-tert-butylphosphino)ferrocene palladiumdichloride (0.033 g, 0.041 mmol), and the mixture was degassed (3×;vacuum/nitrogen). The reaction was immersed in an oil bath at 85° C. andstirred for 1 h. The temperature was reduced to 95° C., and the reactionwas stirred overnight. The reaction mixture was diluted with ethylacetate, washed with water, and washed with brine. The organic layer wascollected, and the aqueous layers were sequentially extracted with ethylacetate (3×). The combined organic layers were dried over anhydroussodium sulfate and concentrated under reduced pressure. The resultingresidue was purified by ISCO flash chromatography (12 g column; 0-20%methanol in dichloromethane) to give tert-butyl4-(4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)piperidine-1-carboxylate(0.138 g, 0.318 mmol, 79% yield) as a light tan solid.

Example 13. tert-Butyl(R)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrolidine-1-carboxylate

13A. tert-Butyl(R)-2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrollidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (1.1 g, 4.62 mmol) and(R)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (1.38 g, 6.01mmol) in ethyl acetate (24 mL) at room temperature was added pyridine(1.87 mL, 23.1 mmol) followed by dropwise addition of1-propanephosphonic anhydride (50 wt % in DMF) (5.50 mL, 9.24 mmol).During the addition, a small amount of gum formed but overtime itdissolved. The reaction mixture stirred for 2.5 h and was diluted withethyl acetate, washed with 1 N aqueous sodium hydroxide (2×), washedwith 10% lithium chloride (2×), and washed with brine. The organic layerwas collected, and the aqueous layers were sequentially extracted withethyl acetate (3×). The combined organic layers were dried overanhydrous magnesium sulfate and concentrated under reduced pressure togive tert-butyl(R)-2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrollidine-1-carboxylate(2.13 g, 4.74 mmol, 100% yield) as a tan foam. LC/MS [M+H]⁺=449.4 and451.4. ¹H-NMR (400 MHz, CDCl₃) δ 8.85-8.62 (m, 1H), 8.56-8.47 (m, 1H),8.15-8.08 (m, 1H), 7.66 (d, J=9.0 Hz, 1H), 7.57-7.49 (m, 1H), 5.74-5.46(m, 2H), 4.85-4.60 (m, 1H), 4.41-4.26 (m, 1H), 3.42 (br d, J=6.8 Hz,1H), 2.84 (br dd, J=14.2, 5.2 Hz, 1H), 2.72-2.59 (m, 1H), 2.24-2.07 (m,1H), 2.03-1.83 (m, 3H), and 1.82-1.57 (m, 9H).

13B. tert-Butyl(R)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

A solution of tert-butyl(R)-2-(2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate(2.1 g, 4.67 mmol) and sodium hydroxide (1.87 g, 46.7 mmol) in EtOH (33mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 1 h under a condensor of nitrogen and was concentrated underreduced pressure. The residue was dissolved in ethyl acetate and water.The organic layer was collected and washed with water and brine. Theaqueous layers were sequentially extracted with ethyl acetate (3×), andthe combined organic layer was dried over anhydrous magnesium sulfate.Concentration under reduced pressure afforded tert-butyl(R)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(1.84 g, 4.27 mmol, 91% yield) as a light amber foam. LC/MS [M+H]⁺=431.4and 433.2.

13C. tert-Butyl(R)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methylpyrrolidine-1-carboxylate

A solution of tert-butyl(R)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(1.84 g, 4.27 mmol) and mCPBA (1.963 g, 8.53 mmol) in dichloromethane(23 mL) was stirred at room temperature for 3 h. To the reaction mixturewas added ammonium hydroxide (7.6 mL) dropwise followed by tosyl-Cl(1.63 g, 8.53 mmol) at 0° C. The resulting reaction mixture was stirredfor 60 min. The homogeneous reaction mixture was extracted withdichloromethane (3×) and the organic layer was collected. The aqueouslayer was diluted with water, the pH was adjusted to ˜5 with 6N aqueoushydrochloric acid and extracted with ethyl acetate (3×). The combinedorganic layers were dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue was purified by ISCOflash silica gel chromatography (80 g column; 0%-10% methanol indichloromethane) to afford tert-butyl(R)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methylpyrrolidine-1-carboxylate(0.36 g, 0.81 mmol, 20% yield) as an amber foam. LC/MS [M+H]⁺=446.4 and448.4.

Example 13

A mixture of tert-butyl(R)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(0.7 g, 1.568 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.609 g,3.14 mmol), and tripotassium phospate (2M in water) (2.35 ml, 4.70 mmol)in dioxane (9.6 mL) was degassed (3×; vacuum/nitrogen). To the mixturewas added 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride(0.128 g, 0.157 mmol), and the mixture degassed (3×; vacuum/nitrogen).The reaction was immersed in an oil bath at 80° C. and stirredovernight. The reaction mixture was diluted with ethyl acetate andwashed with water and brine. The organic layer was collected and theaqueous layers were sequentially extracted with ethyl acetate (3×). Thecombined organic layers were dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. The residue was purified by ISCOflash chromatography (40 g; 0-25% methanol in dichloromethane). A smallsample was purified via preparative LC/MS with the following conditions.Column: XBridge C18, 200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: a 0-minutehold at 11% B, 11-51% B over 20 minutes, then a 4-minute hold at 100% B;Flow Rate: 20 mL/min; Column Temperature: 25° C. Fraction collection wastriggered by MS signals. Fractions containing the desired product werecombined and dried via centrifugal evaporation to give tert-butyl(R)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyyrolidine-1-carboxylate(0.500 g, 1.15 mmol, 74% yield) as a gold solid.

Example 14.(R)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(R)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(0.5 g, 1.15 mmol, Example 4) and TFA (2.88 ml) was stirred at roomtemperature for 60 min. The TFA was removed under reduced pressure, andthe residue was azeotroped with dioxane (3×). A 27 mg sample waspurified via preparative LC/MS with the following conditions. Column:XBridge C18, 200 mm×19 mm, 5 m particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Gradient: a 5-minutehold at 0% B, 0-20% B over 20 minutes, then a 5-minute hold at 100% B;Flow Rate: 20 mL/min; Column Temperature: 25° C. Fraction collection wastriggered by MS signals. Fractions containing the desired product werecombined and dried via centrifugal evaporation to give(R)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine(8.9 mg, 0.027 mmol).

Example 15.(R)-1-(2-((4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidin-1-yl)propane-1-one

To a mixture of(R)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine(0.02 g, 0.060 mmol), propionic acid (6.72 μl, 0.090 mmol), and hunig'sbase (0.105 mL, 0.600 mmol) in N,N-dimethylformamide (1.9 mL) was added1-propanephosphonic anhydride (50 wt % in DMF) (0.039 mL, 0.066 mmol).The reaction was placed on a shaker plate for 60 min. The reactionmixture was filtered through a Whatman 0.45 um PTFE w/GMF filter disc.The crude material was purified via preparative LC/MS with the followingconditions. Column: XBridge C18, 200 mm×19 mm, 5 m particles; MobilePhase A: 5:95 acetonitrile:water with 0.1% trifluoroacetic acid; MobilePhase B: 95:5 acetonitrile:water with 0.1% trifluoroacetic acid;Gradient: a 1-minute hold at 0% B, 0-40% B over 25 minutes, then a6-minute hold at 100% B; Flow Rate: 20 mL/min; Column Temperature: 25°C. Fraction collection was triggered by MS signals. Fractions containingthe desired product were combined and dried via centrifugal evaporationto(R)-1-(2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidin-1-yl)propane-1-one(4 mg, 17% yield).

Example 16. tert-Butyl(S)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyyrolidine-1-carboxylate

16A. tert-Butyl(S)-2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrollidine-1-carboxylate

To a suspension of 7-bromoquinoline-3,4-diamine (2.0 g, 8.40 mmol) and(S)-2-(1-(tert-butoxycarbonyl)pyrrolidin-2-yl)acetic acid (2.22 g, 9.66mmol) in ethyl acetate (43 mL) at room temperature was added pyridine(3.40 mL, 42.0 mmol) followed by dropwise (to minimize gum formation)addition of 1-propanephosphonic anhydride (50 wt % in DMF) (10.00 mL,16.8 mmol). The reaction mixture stirred for 3 h and was diluted withethyl acetate, washed with 1 N aqueous sodium hydroxide (2×), 10%lithium chloride (2×), and brine. The organic layer was collected, andthe aqueous layers were sequentially extracted with ethyl acetate (3×).The combined organic layers were dried over anhydrous magnesium sulfateand concentrated under reduced pressure to give tert-butyl(S)-2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrollidine-1-carboxylate(4.2 g, 9.35 mmol, 100% yield) as a tan foam. LC/MS [M+H]⁺=449.4 and451.4.

16B. tert-Butyl(S)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

A solution of tert-butyl(S)-2-(2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate(3.77 g, 8.39 mmol) and sodium hydroxide (3.36 g, 84 mmol) in EtOH (60mL) was immersed in an oil bath at 80° C. The reaction mixture washeated for 2.5 h under a condensor of nitrogen and was concentratedunder reduced pressure. The residue was dissolved in ethyl acetate andwater. The organic layer was collected and washed with water and brine.The aqueous layers were sequentially extracted with ethyl acetate (3×),and the combined organic layer was dried over anhydrous magnesiumsulfate. Concentration under reduced pressure afforded tert-butyl(S)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(3.34 g, 7.74 mmol, 92% yield) as a light amber foam. LC/MS [M+H]⁺=431.2and 433.2.

16C. tert-Butyl(S)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methylpyrrolidine-1-carboxylate

A clear solution of tert-butyl(S)-2-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(3.34 g, 7.74 mmol) and mCPBA (3.56 g, 15.49 mmol) in dichloromethane(42 mL) was stirred at room temperature for 4 h. To the reaction mixturewas added ammonium hydroxide (14 mL) dropwise followed by tosyl-Cl (2.95g, 15.5 mmol) at 0° C. The resulting reaction mixture was stirred for 60min. The homogeneous reaction mixture was extracted with dichloromethane(3×) and the organic layer was collected. The aqueous layer was dilutedwith water, the pH was adjusted to ˜5 with 6N aqueous hydrochloric acidand extracted with ethyl acetate (3×). The combined organic layers weredried over anhydrous magnesium sulfate and concentrated under reducedpressure. The residue was purified by ISCO flash silica gelchromatography (120 g column; 0%-10% methanol in dichloromethane) toafford tert-butyl(S)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methylpyrrolidine-1-carboxylate(2.0 g, 4.48 mmol, 58% yield) as an amber foam. A small sample of thecrude material was purified via preparative LC/MS with the followingconditions. Column: XBridge C18, 200 mm×19 mm, 5 m particles; MobilePhase A: 5:95 acetonitrile:water with 10-mM ammonium acetate; MobilePhase B: 95:5 acetonitrile:water with 10 mM ammonium acetate; Gradient:a 0-minute hold at 20% B, 20-60% B over 20 minutes, then a 4-minute holdat 100% B; Flow Rate: 20 mL/min; Column Temperature: 25° C. Fractioncollection was triggered by MS and UV signals. Fractions containing thedesired product were combined and dried via centrifugal evaporation.LC/MS [M+H]⁺=446.2 and 448.2. ¹H NMR (500 MHz, DMSO-d₆) δ 8.12-7.94 (m,1H), 7.95-7.87 (m, 1H), 7.71-7.67 (m, 1H), 7.39-7.34 (m, 1H), 6.76-6.63(m, 1H), 4.27-4.12 (m, 1H), 3.36-3.06 (m, 3H), 2.95-2.70 (m, 1H),1.97-1.70 (m, 5H), 1.43-1.09 (m, 9H).

Example 16

A mixture of tert-butyl(S)-2-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(0.71 g, 1.59 mmol),3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.617 g,3.18 mmol), and tripotassium phospate (2M in water) (2.39 ml, 4.77 mmol)in dioxane (9.76 ml) was degassed (3×; vacuum/nitrogen). To the mixturewas added 1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride(0.130 g, 0.159 mmol), and the mixture degassed (3×; vacuum/nitrogen).The reaction was immersed in an oil bath at 80° C. and stirredovernight. The reaction mixture was diluted with ethyl acetate andwashed with water and washed with brine. The organic layer was collectedand the aqueous layers were sequentially extracted with ethyl acetate(3×). The combined organic layers were dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by ISCO flash chromatography (40 g; 0-25% methanol indichloromethane). A small sample of the crude material was purified viapreparative LC/MS with the following conditions. Column: XBridge C18,200 mm×19 mm, 5 m particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10-mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10-mM

ammonium acetate; Gradient: a 0-minute hold at 12% B, 12-52% B over 20minutes, then a 4-minute hold at 100% B; Flow Rate: 20 mL/min; ColumnTemperature: 25° C. Fraction collection was triggered by MS and UVsignals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give tert-butyl(S)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyyrolidine-1-carboxylate(0.600 g, 1.38 mmol, 87% yield) as a gold solid.

Example 17.(S)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine

A homogeneous mixture of tert-butyl(S)-2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate(0.60 g, 1.38 mmol) and TFA (3.46 ml) was stirred at room temperaturefor 60 mins. The TFA was removed under reduced pressure, and the residuewas azeotroped with dioxane (3×). The crude material was purified viapreparative LC/MS with the following conditions: Column: XBridge C18,200 mm×19 mm, 5-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 0.1% trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:waterwith 0.1% trifluoroacetic acid; Gradient: a 1-minute hold at 0% B, 0-40%B over 20 minutes, then a 4-minute hold at 100% B; Flow Rate: 20 mL/min;Column Temperature: 25° C. Fraction collection was triggered by MS andUV signals. Fractions containing the desired product were combined anddried via centrifugal evaporation to give(S)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine(8.9 mg, 0.027 mmol).

Example 18.(S)-1-(2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidin-1-yl)propane-1-one

To a mixture of(S)-7-(1H-pyrazol-3-yl)-2-(pyrrolidin-2-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine(0.02 g, 0.060 mmol), butyric acid (0.017 mL, 0.180 mmol), and hunig'sbase (0.105 mL, 0.600 mmol) in N,N-dimethylformamide (1.9 mL) was added1-propanephosphonic anhydride (50 wt % in DMF) (0.071 mL, 0.120 mmol).The reaction was homogeneous and stirred at 50° C. overnight. Thereaction mixture was filtered through a Whatman 0.45 um PTFE w/GMFfilter disc. The crude material was purified via preparative LC/MS withthe following conditions. Column: XBridge C18, 200 mm×19 mm, 5 mparticles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammoniumacetate; Gradient: a 0-minute hold at 4% B, 4-44% B over 20 minutes,then a 4-minute hold at 100% B; Flow Rate: 20 mL/min; ColumnTemperature: 25° C. Fraction collection was triggered by MS signals.Fractions containing the desired product were combined and dried viacentrifugal evaporation to(S)-1-(2-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidin-1-yl)propane-1-one(8.3 mg, 35% yield).

Example 19. Methyl3-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

19A.tert-Butyl3-(2-((4-amino-7-bromoquinolin-3-yl)amino)-2-oxoethyl)pyrrolidine-1-carboxylate

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed 7-bromoquinoline-3,4-diamine (6 g,25.2 mmol, 1 equiv), DIEA (9.8 g, 0.08 mmol, 3 equiv),2-[1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]acetic acid (11.6 g, 0.05mmol, 2.01 equiv) and HATU (14.4 g, 0.04 mmol, 1.5 equiv) in DCM (120mL). The resulting solution was stirred for 2 hr at room temperature.The resulting mixture was concentrated. This resulted in 12 g (105.97%)of tert-butyl3-[[(4-amino-7-bromoquinolin-3-yl)carbamoyl]methyl]pyrrolidine-1-carboxylateas yellow crude oil.

19B. tert-Butyl3-((7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

Into a 500-mL round-bottom flask, was placed tert-butyl3-[[(4-amino-7-bromoquinolin-3-yl)carbamoyl]methyl]pyrrolidine-1-carboxylate(6 g, 13.4 mmol, 1 equiv) and NaOH (5.3 g, 133.53 mmol, 10 equiv) inEtOH (120 mL). The resulting solution was stirred for 30 min at 80° C.The resulting mixture was concentrated. The pH value of the solution wasadjusted to 7 with HCl (12 mol/L). The resulting solution was extractedwith 2×200 mL of dichloromethane, the organic layers was washed with1×200 mL of brine. the organic layers combined. The residue was appliedonto a silica gel column with dichloromethane/methanol (30:1). Thisresulted in 5.5 g (96%) of tert-butyl3-([7-bromo-1H-imidazo[4,5-c]quinolin-2-yl]methyl)pyrrolidine-1-carboxylateas a yellow solid.

19C. tert-Butyl3-((4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

Into a 250-mL round-bottom flask, was placed tert-butyl3-([7-bromo-1H-imidazo[4,5-c]quinolin-2-yl]methyl)pyrrolidine-1-carboxylate(5 g, 11.59 mmol, 1 equiv) and mCPBA (4.0 g, 23.18 mmol, 2.00 equiv) inDCM (100 mL). The resulting solution was stirred for 2 hr at roomtemperature. The resulting mixture was concentrated. The residue wasapplied onto a silica gel column with dichloromethane/methanol (10:1).This resulted in 3.6 g (69%) of7-bromo-2-([1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]methyl)-1H-imidazo[4,5-c]quinolin-5-ium-5-olateas a yellow solid.

Into a 250-mL round-bottom flask, was placed7-bromo-2-([1-[(tert-butoxy)carbonyl]pyrrolidin-3-yl]methyl)-1H-imidazo[4,5-c]quinolin-5-ium-5-olate(2 g, 4.47 mmol, 1 equiv), ammonium hydroxide (10 mL) and TsCl (1.7 g,8.92 mmol, 1.99 equiv) in DCM (20 mL). The resulting solution wasstirred for 2 hr at room temperature. The resulting mixture wasconcentrated. The residue was applied onto a silica gel column withdichloromethane/methanol (20:1). This resulted in 1.6 g (80.18%) oftert-butyl3-([4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl]methyl)pyrrolidine-1-carboxylateas a yellow solid.

19D. tert-Butyl3-((4-amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

Into a 100-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed tert-butyl3-([4-amino-7-bromo-1H-imidazo[4,5-c]quinolin-2-yl]methyl)pyrrolidine-1-carboxylate(1.5 g, 3.36 mmol, 1 equiv), Cs₂CO₃ (3.3 g, 10.08 mmol, 3 equiv),5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.3 g, 6.72mmol, 2 equiv) and Pd(dppf)Cl₂—CH₂Cl₂ (0.5 g, 0.61 mmol, 0.18 equiv) indioxane (30 mL) and H₂O (5 mL). The resulting solution was stirred for16 hr at 100° C. The resulting mixture was concentrated. The residue wasapplied onto a silica gel column with dichloromethane/methanol (10:1).This resulted in 870 mg (60%) of tert-butyl3-[[4-amino-7-(1H-pyrazol-5-yl)-1H-imidazo[4,5-c]quinolin-2-yl]methyl]pyrrolidine-1-carboxylateas a yellow solid

19E.7-(1H-Pyrazol-3-yl)-2-(pyrrolidin-3-ylmethyl)-1H-imidazo[4,5-c]quinolin-4-amine

Into a 50-mL round-bottom flask, was placed tert-butyl3-[[4-amino-7-(1H-pyrazol-5-yl)-1H-imidazo[4,5-c]quinolin-2-yl]methyl]pyrrolidine-1-carboxylate(100 mg, 0.23 mmol, 1 equiv) in hydrogen chloride in dioxane (3 mL, 4N).The resulting solution was stirred for 2 hr at room temperature. Theresulting mixture was concentrated. The crude product was purified byreverse phase, preparative HPLC with the following conditions: Column:Xselect CSH OBD Column 30*150 mm 5 um n; Mobile Phase A: Water (0.05%TFA), Mobile Phase B: ACN; Flow rate: 60 mL/min; Gradient: 5% B to 15% Bin 7 min; 254/210 nm; Rt: 6.95 min, This resulted in 20 mg (26.01%) of7-(1H-pyrazol-5-yl)-2-[(pyrrolidin-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-4-amineas a white solid. LC/MS [M+H]⁺=334.2. ¹H NMR (400 MHz, Methanol-d₄) δ:8.06-8.00 (m, 2H), 8.10 (s, 1H), 7.80-7.66 (m, 2H), 6.76 (s, 1H),3.60-3.30 (m, 1H), 3.25-3.15 (m, 1H), 3.15-3.00 (m, 3H), 2.90-2.70 (m,2H), 2.20-2.05 (m, 1H), and 1.80-1.65 (m, 1H).

Example 19

Into a 25-mL round-bottom flask, was placed7-(1H-pyrazol-5-yl)-2-[(pyrrolidin-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-4-amine (40 mg, 0.12 mmol, 1 equiv),DMF (2 mL), dimethyl carbonate (21.6 mg, 0.24 mmol, 2.00 equiv), K₂CO₃(49.7 mg, 0.36 mmol, 3 equiv). The resulting solution was stirred for 16hrs at 100 degrees C. The resulting mixture was concentrated. The crudeproduct was purified by reverse phase, preparative HPLC with thefollowing conditions: Column, XBridge Prep C18 OBD Column, Sum, 19*150mm; mobile phase, Water (10 MMOL/L NH₄HCO₃) and ACN (13% Phase B up to36% in 7 min); Detector, UV. 12 mg product was obtained. This resultedin 12 mg (26%) of methyl 3-[[4-amino-7-(1H-pyrazol-5-yl)-1H-imidazo[4,5-c]quinolin-2-yl]methyl]pyrrolidine-1-carboxylate as a white solid.

Example 20.1-(3-((4-Amino-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidin-1-yl)ethan-1-one

Into a 50-mL round-bottom flask, was placed7-(1H-pyrazol-5-yl)-2-[(pyrrolidin-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-4-amine(100 mg, 0.30 mmol, 1 equiv), TEA (151.8 mg, 1.50 mmol, 5 equiv) andacetyl acetate (61.2 mg, 0.60 mmol, 2 equiv) in DCM (5 mL). Theresulting solution was stirred for 2 hr at room temperature. Theresulting mixture was concentrated and MeOH (20 mL) was added. Theresulting solution was stirred for 16 h at 80° C. The resulting mixturewas concentrated, and the crude product was purified by reverse phase,preparative HPLC with the following conditions: Column: XBridge Prep C18OBD Column, 5 um, 19*150 mm; Mobile Phase A: Water (10 MMOL/L NH₄HCO₃),Mobile Phase B: ACN; Flow rate: 25 mL/min; Gradient: 5% B to 40% B in7.5 min; 254/210 nm; Rt: 6.6 min. This resulted in 20 mg (18%) of1-(3-[[4-amino-7-(1H-pyrazol-5-yl)-1H-imidazo[4,5-c]quinolin-2-yl]methyl]pyrrolidin-1-yl)ethan-1-oneas a light yellow solid

Example 21.2-((1-Ethylpyrrolidin-3-yl)methyl)-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amine

Into a 50-mL round-bottom flask, was placed7-(1H-pyrazol-5-yl)-2-[(pyrrolidin-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-4-amine(19E, 100 mg, 0.30 mmol, 1 equiv), AcOH (0.1 mL), acetaldehyde (26.4 mg,0.60 mmol, 2.00 equiv) and NaBH₃CN (37.7 mg, 0.60 mmol, 2.00 equiv) inMeOH (3 mL). The resulting solution was stirred for 2 h at roomtemperature. The resulting mixture was concentrated, and the crudeproduct was purified by reverse phase, preparative HPLC with thefollowing conditions: Column: XBridge BEH130 Prep C18 OBD Column 19*150mm 5 um 13 nm; Mobile Phase A: Water (10 MMOL/L NH₄HCO₃), Mobile PhaseB: ACN; Flow rate: 25 mL/min; Gradient: 30% B to 60% B in 8 min; 254/210nm; Rt: 6.95 min. This resulted in 29 mg (27%) of2-[(1-ethylpyrrolidin-3-yl)methyl]-7-(1H-pyrazol-3-yl)-1H-imidazo[4,5-c]quinolin-4-amineas a off-white solid.

Example 22.2-((1-Ethylpyrrolidin-3-yl)methyl)-7-(1H-pyrazol-1-yl)-1H-imidazo[4,5-c]quinolin-4-amine

Into a 25-mL round-bottom flask, was placed7-(1H-pyrazol-1-yl)-2-[(pyrrolidin-3-yl)methyl]-1H-imidazo[4,5-c]quinolin-4-amine (50 mg, 0.15 mmol, 1 equiv), MeOH (2 mL), AcOH (0.2mL), acetaldehyde (9.9 mg, 0.22 mmol, 1.50 equiv), NaBH₃CN (14.1 mg,0.22 mmol, 1.5 equiv). The resulting solution was stirred for 2 h atroom temperature. The resulting mixture was concentrated, and the crudeproduct was purified by reverse phase, preparative HPLC with thefollowing conditions: Column, XBridge Prep C18 OBD Column, Sum, 19*150mm; mobile phase, Water (10 MMOL/L NH₄HCO₃) and ACN (20% PhaseB up to22% in 10 min); Detector, UV. This resulted in 20 mg (37%) of2-[(1-ethylpyrrolidin-3-yl) methyl]-7-(1H-pyrazol-1-yl)-1H-imidazo [4,5-c]quinolin-4-amine as a white solid.

Example 23. tert-Butyl3-((4-amino-7-(1H-pyrazol-1-yl)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)pyrrolidine-1-carboxylate

Into a 25-mL round-bottom flask, was placed7-(1H-pyrazol-1-yl)-2-[(pyrrolidin-3-yl) methyl]-1H-imidazo [4,5-c]quinolin-4-amine (150 mg, 0.45 mmol, 1 equiv), DCM (4 mL), methylcarbonochloridate (85.0 mg, 0.90 mmol, 2.00 equiv), TEA (136.6 mg, 1.35mmol, 3 equiv). The resulting solution was stirred for 2 hrs at roomtemperature. The resulting mixture was concentrated. Then the crudeproduct was added in MeOH (2 mL), NaOH (13.3 mg, 0.33 mmol, 2.99 equiv).The resulting solution was stirred for 2 hrs at 80° C. The resultingmixture was concentrated. The crude product was purified by reversephase, preparative HPLC with the following conditions: Column: XBridgePrep C₁₈ OBD Column 19*150 mm Sum; Mobile Phase A: Water (10 MMOL/LNH₄HCO₃+0.1% NH₃·H₂O), Mobile Phase B: ACN; Flow rate: 25 mL/min;Gradient: 15% B to 45% B in 7.5 min; 254/210 nm; Rt: 6.08 min. Thisresulted in 20 mg (46%) of methyl3-[[4-amino-7-(1H-pyrazol-1-yl)-1H-imidazo[4,5-c]quinolin-2-yl]methyl]pyrrolidine-1-carboxylateas a white solid.

Starting with the appropriate starting material, Examples 24 through 40were prepared in a similar fashion as described in the procedures forExample 3; Examples 41 through 50 were prepared in a similar fashion asdescribed in the procedures for Example 4; Examples 51 through 59 wereprepared in a similar fashion as described in the procedures for Example5; Examples 60 through 70 were prepared in a similar fashion asdescribed in the procedures for Example 8; Examples 71 through 76 wereprepared in a similar fashion as described in the procedures for Example9; and Example 77 was prepared in a similar fashion as described in theprocedures for Example 11. Examples 78 through 85 were prepared in asimilar fashion as described in the procedures for Example 15. Examples86 through 8 were prepared in a similar fashion as described in theprocedures for Example 15.

Biological data of compounds that were assayed using one or more of theabove procedures. Unless otherwise indicated, the TLR7 agonist EC₅₀ andTLR8 agonist EC₅₀ of the below compounds were measured at values >100μM.

LC/MS [M + H]⁺/ RT(Method)/ ¹H NMR Ex NLRP3 (500 MHz, DMSO-d₆, unlessNo. Structure hIL1β EC₅₀ otherwise indicated) 1

434.4/ 0.71 min (C)/ 0.33 μM TLR8 EC₅₀ 10 μM δ 8.05-7.99 (m, 1H), 7.95(s, 1H), 7.73-7.63 (m, 2H), 6.76- 6.71 (m, 1H), 4.40-4.01 (m, 1H),3.94-3.83 (m, 1H), 3.51- 3.36 (m, 1H), 3.03-2.92 (m, 1H), 2.84 (br t, J= 11.4 Hz, 1H), 2.21-2.09 (m, 1H), 1.89-1.73 (m, 2H), 1.54-1.42 (m, 1H),1.42-1.26 (m, 9H) 2

334.3/ 0.50 min (C)/ 0.51 μM δ 8.30-8.15 (m, 2H), 8.04- 7.97 (m, 1H),7.91-7.80 (m, 1H), 6.89-6.81 (m, 1H), 3.60- 3.49 (m, 1H), 3.09-2.96 (m,1H), 2.59-2.53 (m, 2H), 2.31- 2.23 (m, 1H), 1.99-1.71 (m, 3H), 1.21-1.08(m, 1H) 3

427.3/ 0.95 min (A)/ 0.98 μM TLR7 EC₅₀ 0.62 μM δ 8.23-8.10 (m, 2H),8.06- 7.89 (m, 1H), 7.82 (br s, 1H), 6.85 (d, J = 2.1 Hz, 1H), 4.58 (brd, J = 11.1 Hz, 1H), 4.25 (br s, 1H), 3.88 (br s, 2H), 2.92 (br s, 1H),2.57-2.53 (m, 1H), 2.37- 2.21 (m, 1H), 1.95 (br s, 1H), 1.72-1.60 (m,2H), 1.52 (br s, 2H), 1.15 (t, J = 7.2 Hz, 1H) 4

418.1/ 1.00 min (A)/ 0.26 μM δ 8.12 (br d, J = 8.5 Hz, 1H), 8.03 (s,1H), 7.73 (br s, 2H), 6.80 (d, J = 1.5 Hz, 1H), 6.51 (br s, 1H), 4.64(br d, J = 7.6 Hz, 2H), 4.50 (br d, J = 10.7 Hz, 1H), 4.08 (br d, J =14.0 Hz, 1H), 3.96 (br d, J = 13.7 Hz, 1H), 3.11 (br s, 1H), 2.63 (br s,1H), 2.49- 2.34 (m, 2H), 2.24-2.03 (m, 1H), 1.98 (br d, J = 11.3 Hz,1H), 1.87 (br s, 1H), 1.79 (br d, J = 15.0 Hz, 1H), 1.53-1.42 (m, 3H),1.02 (dt, J = 15.6, 7.6 Hz, 3H) 5

432.0/ 1.24 min (A)/ 0.33 μM δ 8.23 (br d, J = 8.6 Hz, 1H), 8.04 (s,1H), 7.93 (br s, 1H), 7.74 (br s, 2H), 6.80 (d, J = 1.9 Hz, 1H), 4.64(br d, J = 13.4 Hz, 1H), 3.94 (br d, J = 13.6 Hz, 1H), 3.74 (br s, 3H),3.47 (br s, 1H), 3.24-3.06 (m, 1H), 2.49-2.29 (m, 2H), 2.10 (br s, 1H),1.84 (br d, J = 12.3 Hz, 1H), 1.73 (br s, 6H), 1.56 (br s, 1H), 1.06-0.96 (m, 3H) 6

434.2/ 1.48 min (B)/ 0.36 μM TLR8 EC₅₀ 10 μM δ 8.05-7.99 (m, 1H), 7.95(s, 1H), 7.73-7.63 (m, 2H), 6.76- 6.71 (m, 1H), 4.40-4.01 (m, 1H),3.94-3.83 (m, 1H), 3.51- 3.36 (m, 1H), 3.03-2.92 (m, 1H), 2.84 (br t, J= 11.4 Hz, 1H), 2.21-2.09 (m, 1H), 1.89-1.73 (m, 3H), 1.54-1.42 (m, 1H),1.42-1.26 (m, 9H) 7

334.0/ 0.99 min (B)/ 1.73 μM δ 8.30-8.15 (m, 2H), 8.04- 7.97 (m, 1H),7.91-7.80 (m, 1H), 6.89-6.81 (m, 1H), 3.60- 3.49 (m, 1H), 3.09-2.96 (m,1H), 2.59-2.53 (m, 2H), 2.31- 2.23 (m, 1H), 1.99-1.71 (m, 3H), 1.21-1.08(m, 1H) 8

416.3/ 1.24 min (B)/ 1.9 μM δ 8.11-8.04 (m, 1H), 8.01- 7.95 (m, 1H),7.77-7.66 (m, 2H), 6.83-6.72 (m, 1H), 3.71- 3.52 (m, 4H), 3.10-2.92 (m,1H), 2.79-2.61 (m, 1H), 2.31- 2.01 (m, 5H), 1.96-1.93 (m, 1H), 1.89-1.68(m, 3H), 1.58- 1.34 (m, 1H) 9

376.1/ 0.76 min (A)/ 6.6 μM δ 8.21 (br d, J = 6.4 Hz, 2H), 8.00 (br s,1H), 7.85 (br s, 1H), 6.83 (br s, 1H), 4.03-3.87 (m, 1H), 3.87-3.76 (m,1H), 3.71 (br d, J = 10.4 Hz, 1H), 3.66- 3.56 (m, 1H), 2.56-2.53 (m,2H), 2.38 (br d, J = 8.2 Hz, 1H), 2.30 (q, J = 7.4 Hz, 2H), 1.06- 0.96(m, 3H) 10

420.4/ 0.92 min (A)/ 0.75 μM (400 MHz, METHANOL-d₄) δ 8.08 (d, J = 8.3Hz, 1H), 8.01 (s, 1H), 7.82 (br s, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.52(s, 1H), 6.74 (d, J = 2.0 Hz, 1H), 3.95 (br d, J = 8.4 Hz, 1H),3.80-3.60 (m, 3H), 3.51 (br d, J = 9.6 Hz, 1H), 2.54-2.28 (m, 2H), 1.48(s, 9H) 11

390.2/ 1.08 min (A)/ 7.0 μM δ 8.12-8.03 (m, 1H), 8.02- 7.95 (m, 1H),7.75-7.64 (m, 2H), 6.80-6.69 (m, 1H), 3.95- 3.84 (m, 2H), 3.36-3.26 (m,1H), 2.82-2.70 (m, 1H), 1.82- 1.70 (m, 6H), 1.13-0.98 (m, 4H) 12

434.4/ 0.70 min (D)/ 4.9 μM 13

434.2/ 1.42 min (B)/ 1.52 μM δ 9.09-8.44 (m, 1H), 8.25- 8.11 (m, 2H),8.04-7.94 (m, 1H), 7.90-7.81 (m, 1H), 6.87- 6.78 (m, 1H), 4.31-4.14 (m,1H), 3.54-3.41 (m, 2H), 3.40- 3.21 (m, 2H), 3.20-3.04 (m, 1H), 3.02-2.81(m, 1H), 2.03- 1.74 (m, 5H), 1.41-1.30 (m, 9H) 14

334.0/ 0.95 min (B)/ 0.11 μM δ 8.96-8.75 (m, 2H), 8.23- 8.07 (m, 2H),7.97 (br d, J = 7.9 Hz, 1H), 7.86-7.76 (m, 1H), 6.86-6.77 (m, 1H),4.07-3.93 (m, 1H), 3.53-3.17 (m, 4H), 2.25-2.11 (m, 1H), 2.05-1.84 (m,2H), 1.77-1.65 (m, 1H) 15

390.1/ 1.13 min (B)/ 1.8 μM δ 8.22-8.08 (m, 2H), 8.01- 7.93 (m, 1H),7.87-7.77 (m, 1H), 6.87-6.79 (m, 1H), 4.48- 4.36 (m, 1H), 3.63-3.46 (m,3H), 3.45-3.31 (m, 1H), 2.97- 2.83 (m, 2H), 2.43-2.18 (m, 2H), 1.94-1.73(m, 4H), 1.15 (t, J = 7.2 Hz, 1H), 1.02-0.92 (m, 3H) 16

434.2/ 1.6 min (B)/ 3.7 μM δ 8.07-7.91 (m, 2H), 7.75- 7.66 (m, 2H),6.80-6.71 (m, 1H), 4.29-4.13 (m, 1H), 3.64- 3.47 (m, 1H), 3.38-3.21 (m,2H), 2.94-2.78 (m, 1H), 1.98- 1.71 (m, 8H), 1.45-1.12 (m, 9H) 17

334.0/ 1.5 min (B)/ 1.2 μM δ 8.24-8.09 (m, 2H), 8.00- 7.88 (m, 1H),7.87-7.74 (m, 1H), 6.87-6.77 (m, 1H), 4.11- 3.99 (m, 1H), 3.45-3.20 (m,1H), 2.96-2.86 (m, 1H), 2.76- 2.67 (m, 1H), 2.28-2.12 (m, 1H), 2.09-1.86(m, 3H), 1.82- 1.69 (m, 1H) 18

362.2/ 1.01 min (B)/ 4.9 μM δ 8.11-7.96 (m, 2H), 7.79- 7.68 (m, 2H),6.81-6.75 (m, 1H), 4.50-4.35 (m, 1H), 3.60- 3.39 (m, 4H), 3.23-2.95 (m,1H), 2.91-2.76 (m, 1H), 2.39- 2.19 (m, 2H), 1.95-1.76 (m, 5H), 1.62-1.48(m, 2H), 0.94- 0.86 (m, 3H), 0.80-0.73 (m, 1H) 19

392.2/ 1.76 min (C)/ 1.6 μM (300 MHz, Methanol-d₄) δ 8.06 (d, J = 8.9Hz, 2H), 7.76 (d, J = 24.1 Hz, 2H), 6.78 (s, 1H), 3.74- 3.52 (m, 5H),3.49-3.39 (m, 1H), 3.24-3.16 (m, 1H), 3.14- 3.02 (m, 2H), 2.91-2.74 (m,1H), 2.15 (s, 2H), 1.90-1.71 (m, 1H) 20

376.2/ 1.12 min (C)/ 4.9 μM (DMSO-d₆, 400 MHz, ppm): δ: 13.1 (s, 1H),8.05 (s, 1H), 8.01 (s, 1H), 7.80-7.60 (m, 2H), 6.76 (s, 1H), 6.50-6.40(m, 2H), 3.70-3.10 (m, 7H), 2.90- 2.60 (m, 1H), 2.20-2.00 (m, 1H), 1.95(s, 3H), 1.85-1.60 (m, 1H) 21

362.2/ 1.01 min (C)/ 0.88 μM (CD₃OD, 300 MHz, ppm): δ: 8.10-8.00 (m,2H), 7.80-7.60 (m, 2H), 6.77 (s, 1H), 3.10- 3.00 (m, 2H), 3.00-2.50 (m,6H), 2.46-2.30 (m, 1H), 2.20- 2.00 (m, 1H), 1.75-1.55 (m, 1H), 1.20-1.00(m, 3H) 22

362.2/ 0.82 min (C)/ 1.4 μM (400 MHz, Methanol-d₄) δ 8.43- 8.30 (m, 1H),8.16-8.02 (m, 1H), 7.98 (t, J = 1.9 Hz, 1H), 7.83-7.69 (m, 2H), 6.59 (t,J = 2.1 Hz, 1H), 3.12-3.03 (m, 2H), 3.03-2.97 (m, 1H), 2.97-2.79 (m,2H), 2.78-2.57 (m, 3H), 2.57-2.48 (m, 1H), 2.27-2.02 (m, 1H), 1.81-1.60(m, 1H), 1.18 (t, J = 7.2 Hz, 3H) 23

392.2/ 1.01 min (C)/ 2.2 μM (400 MHz, Methanol-d₄) δ 8.34 (d, J = 2.6Hz, 1H), 8.11 (s, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.84-7.70 (m, 2H), 6.60(t, J = 2.2 Hz, 1H), 3.69-3.54 (m, 5H), 3.52-3.11 (m, 2H), 3.22 (t, J =9.2 Hz, 2H), 2.84 (d, J = 8.3 Hz, 1H), 2.16 (s, 1H), 1.81 (d, J = 7.6Hz, 1H) 24

376.1/ 0.82 min (A)/ 0.77 μM δ 8.23-8.14 (m, 2H), 7.99 (br d, J = 7.3Hz, 1H), 7.83 (br s, 1H), 6.84 (s, 1H), 4.88-4.66 (m, 1H), 4.39-4.18 (m,1H), 3.89-3.84 (m, 1H), 3.20-3.06 (m, 2H), 3.01 (br s, 1H), 2.25 (br s,1H), 2.06 (br d, J = 7.9 Hz, 4H), 1.95-1.82 (m, 2H) 25

390.0/ 0.91 min (A)/ 1.1 μM δ 8.25-8.18 (m, 2H), 8.01 (br d, J = 7.0 Hz,1H), 7.85 (br s, 1H), 6.84 (br s, 1H), 4.81 (br d, J = 13.7 Hz, 1H),3.95-3.88 (m, 1H), 3.45 (br s, 1H), 3.17 (s, 1H), 3.12-2.99 (m, 1H),2.44- 2.35 (m, 2H), 2.26 (br s, 1H), 1.97-1.83 (m, 2H), 1.60 (br s, 1H),1.01 (br d, J = 5.5 Hz, 3H) 26

404.2/ 0.99 min (A)/ 1.2 μM δ 8.09 (br d, J = 7.3 Hz, 1H), 8.02 (br s,1H), 7.80-7.68 (m, 2H), 6.79 (s, 1H), 4.90-4.67 (m, 1H), 4.22 (br s,1H), 4.10- 3.92 (m, 1H), 3.13 (br s, 1H), 2.94 (br s, 1H), 2.89 (s, 1H),2.73 (s, 1H), 2.24 (br s, 1H), 1.80 (br s, 1H), 1.55 (br s, 1H), 1.49(br s, 1H), 1.09-0.91 (m, 6H) 27

404.0/ 1.01 min (A)/ 0.89 μM δ 8.25-8.18 (m, 2H), 8.00 (br d, J = 7.3Hz, 1H), 7.85 (br s, 1H), 6.84 (s, 1H), 4.89-4.71 (m, 1H), 4.45-4.13 (m,1H), 4.03-3.83 (m, 1H), 3.57-3.33 (m, 1H), 3.23-2.97 (m, 1H), 2.35 (brs, 2H), 2.26 (br s, 1H), 1.98-1.84 (m, 2H), 1.59-1.46 (m, 3H), 0.93-0.86(m, 3H) 28

418.1/ 1.26 min (A)/ 1.2 μM TLR8 EC₅₀ 47 μM δ 8.06 (br d, J = 8.2 Hz,1H), 7.98 (s, 1H), 7.69 (br d, J = 8.2 Hz, 2H), 6.77 (s, 1H), 4.67 (brd, J = 11.3 Hz, 1H), 4.35 (br d, J = 12.2 Hz, 1H), 3.10-2.96 (m, 1H),2.91 (br d, J = 18.3 Hz, 1H), 2.25 (br d, J = 11.6 Hz, 1H), 1.97-1.87(m, 2H), 1.85 (br s, 1H), 1.54 (br d, J = 11.9 Hz, 1H), 1.25 (s, 9H) 29

402.1/ 1.15 min (A)/ 2.4 μM δ 8.05 (br d, J = 8.2 Hz, 1H), 8.01-7.93 (m,1H), 7.75-7.66 (m, 2H), 6.77 (s, 1H), 4.77 (br s, 1H), 4.60-4.44 (m,1H), 4.30 (br s, 1H), 3.27-3.07 (m, 1H), 2.25 (br s, 1H), 2.08 (br d, J= 18.3 Hz, 1H), 1.98 (br s, 1H), 1.79 (br s, 1H), 1.59 (br s, 1H), 1.50(br s, 1H), 0.76 (br s, 4H) 30

416.0/ 1.21 min (A)/ 0.68 μM δ 8.21 (s, 1H), 8.18 (br s, 1H), 7.98 (brd, J = 7.0 Hz, 1H), 7.84 (br s, 1H), 6.84 (br s, 1H), 4.75 (br d, J =12.8 Hz, 1H), 3.49 (br s, 1H), 3.46-3.35 (m, 1H), 3.13-2.99 (m, 2H),2.93-2.87 (m, 1H), 2.28-2.05 (m, 4H), 1.95-1.82 (m, 2H), 1.74 (br s,1H), 1.51 (br d, J = 12.2 Hz, 1H), 1.25 (br d, J = 6.4 Hz, 2H) 31

438.2/ 1.06 min (A)/ 1.5 μM δ 8.11 (br s, 1H), 8.03 (br s, 1H), 7.74 (brs, 2H), 7.42 (br s, 5H), 6.79 (br s, 1H), 4.96-4.72 (m, 1H), 3.89 (s,1H), 3.47 (br s, 1H), 3.17 (s, 2H), 3.08-2.90 (m, 1H), 2.29 (br s, 1H),1.81 (br s, 1H), 1.64 (br s, 1H) 32

452.4/ 1.14 min (A)/ 2.5 μM δ 8.19 (br s, 2H), 7.99 (br s, 1H), 7.83 (brs, 1H), 6.83 (br s, 1H), 4.65 (s, 1H), 4.32 (br d, J = 9.5 Hz, 1H),4.17-3.97 (m, 1H), 3.84-3.69 (m, 1H), 3.64- 3.23 (m, 2H), 3.16 (s, 1H),3.14- 3.04 (m, 1H), 3.05-2.92 (m, 1H), 2.81 (br s, 1H), 2.25 (br s, 1H),1.91 (br d, J = 13.1 Hz, 2H), 1.54 (br s, 1H) 33

438.2/ 1.09 min (A)/ 1.0 μM δ 8.23 (br s, 2H), 8.02 (br s, 1H), 7.86 (brs, 1H), 6.84 (br s, 1H), 4.84-4.76 (m, 1H), 4.48- 4.21 (m, 1H), 4.14 (brt, J = 13.9 Hz, 1H), 3.36-3.07 (m, 1H), 2.82 (s, 1H), 2.32 (br s, 1H),2.01 (br s, 1H), 1.98-1.82 (m, 3H), 1.73 (br s, 1H), 1.54 (br s, 1H) 34

406.4/ 0.92 min (A)/ 2.2 μM δ 8.08 (br s, 1H), 8.01 (br s, 1H), 7.73 (brs, 2H), 6.77 (s, 1H), 4.84-4.67 (m, 1H), 4.26- 4.03 (m, 3H), 3.92-3.77(m, 1H), 3.31 (br d, J = 6.1 Hz, 1H), 3.05 (br d, J = 13.1 Hz, 1H), 2.99(s, 1H), 2.25 (br s, 1H), 1.98- 1.88 (m, 2H), 1.85 (br s, 1H), 1.61 (brs, 1H), 1.50 (br s, 1H) 35

420.2/ 0.87 min (A)/ 1.8 μM δ 8.19-8.11 (m, 2H), 7.97 (br d, J = 8.2 Hz,1H), 7.81 (br s, 1H), 6.85 (br s, 1H), 4.76-4.60 (m, 1H), 4.24-4.05 (m,1H), 3.88 (br d, J = 8.2 Hz, 3H), 3.55 (br s, 1H), 3.57-3.42 (m, 1H),3.23-3.11 (m, 3H), 3.03-2.94 (m, 1H), 2.67-2.57 (m, 2H), 2.22 (br s,1H), 1.89 (br d, J = 13.1 Hz, 1H), 1.64-1.37 (m, 1H) 36

470.2/ 1.13 min (A)/ 2.8 μM δ 8.07 (br d, J = 7.9 Hz, 1H), 8.00 (s, 1H),7.96 (s, 1H), 7.75- 7.68 (m, 2H), 6.77 (d, J = 1.8 Hz, 1H), 4.80-4.54(m, 1H), 4.16 (s, 1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.55 (s, 1H), 2.26(br d, J = 11.0 Hz, 1H), 1.91 (s, 1H), 1.59 (br s, 2H), 1.39-1.30 (m,2H), 1.30-1.18 (m, 2H) 37

441.2/ 1.07 min (A)/ 4.7 μM δ 8.06 (br s, 1H), 7.99 (s, 1H), 7.77-7.67(m, 2H), 6.77 (d, J = 2.1 Hz, 1H), 4.71 (br d, J = 8.2 Hz, 1H), 3.69 (brd, J = 14.0 Hz, 1H), 3.23 (br s, 1H), 3.08 (br s, 1H), 2.89 (s, 1H),2.85-2.65 (m, 1H), 2.60 (br s, 2H), 2.54 (s, 2H), 2.28 (br s, 1H), 2.20-2.06 (m, 1H), 1.97-1.85 (m, 2H), 1.68 (br s, 1H) 38

469.1/ 1.26 min (A)/ 2.0 μM TLR8 EC₅₀ 17 μM δ 8.39-8.19 (m, 2H), 8.01(br d, J = 8.2 Hz, 1H), 7.85 (br s, 1H), 6.85 (d, J = 2.2 Hz, 1H),4.89-4.56 (m, 1H), 4.51-4.21 (m, 1H), 3.00 (s, 1H), 2.30 (br s, 1H),2.05 (br d, J = 13.0 Hz, 3H), 1.95 (br s, 2H), 1.77 (br d, J = 11.2 Hz,3H), 1.73-1.61 (m, 3H), 1.53 (br d, J = 11.8 Hz, 3H), 1.30-1.13 (m, 1H)39

455.3/ 1.26 min (C)/ 2.5 μM TLR7 EC₅₀ 35 μM δ 8.05 (br s, 1H), 7.99 (s,1H), 7.71 (br s, 2H), 6.76 (s, 1H), 4.80-4.64 (m, 1H), 4.58-4.35 (m,1H), 4.07 (br d, J = 4.0 Hz, 1H), 2.99 (s, 1H), 2.54 (s, 12H), 2.38 (brd, J = 18.3 Hz, 1H), 2.34- 2.18 (m, 4H), 1.96 (br s, 1H), 1.90 (s, 1H),1.74 (br s, 2H), 1.69 (br s, 3H) 40

452.2/ 1.23 min (A)/ 3.8 μM TLR7 EC₅₀ 58 μM δ 8.04 (br s, 1H), 7.98 (s,1H), 7.70 (br s, 2H), 7.33-7.19 (m, 5H), 6.76 (s, 1H), 4.77 (br d, J =9.8 Hz, 1H), 4.26 (br d, J = 11.9 Hz, 1H), 4.00 (br d, J = 13.4 Hz, 1H),3.85-3.74 (m, 2H), 3.07 (br d, J = 11.6 Hz, 1H), 2.96-2.89 (m, 1H), 2.19(br s, 1H), 1.96-1.87 (m, 1H), 1.74 (br d, J = 14.6 Hz, 1H), 1.56- 1.38(m, 1H) 41

404.1/ 0.91 min (A)/ 0.77 μM δ 8.13 (dd, J = 8.2, 4.3 Hz, 1H), 8.04 (s,1H), 7.95 (s, 1H), 7.81- 7.68 (m, 2H), 6.80 (d, J = 1.8 Hz, 1H), 6.55(br s, 1H), 4.63 (br dd, J = 12.7, 8.4 Hz, 2H), 4.46 (br d, J = 11.6 Hz,1H), 4.04 (br d, J = 11.9 Hz, 1H), 3.91 (br d, J = 13.4 Hz, 1H), 3.30(br s, 1H), 3.22-3.05 (m, 1H), 2.98-2.79 (m, 1H), 2.63 (br t, J = 11.4Hz, 1H), 2.14-2.04 (m, 2H), 1.85 (br d, J = 5.8 Hz, 1H), 1.78 (br d, J =12.5 Hz, 1H), 1.67 (br d, J = 12.2 Hz, 1H), 1.52-1.43 (m, 3H) 42

432.1/ 1.12 min (A)/ 0.22 μM δ 8.30-8.08 (m, 1H), 8.04 (s, 1H), 7.74 (brs, 2H), 6.80 (d, J = 1.8 Hz, 1H), 6.55 (br s, 1H), 4.63 (br d, J = 7.0Hz, 2H), 4.50 (br d, J = 11.0 Hz, 1H), 4.10 (br d, J = 15.0 Hz, 1H),3.97 (br d, J = 14.0 Hz, 1H), 3.10 (br d, J = 11.9 Hz, 1H), 2.86 (br s,1H), 2.64 (br s, 1H), 2.36 (br d, J = 7.3 Hz, 2H), 2.12 (br d, J = 11.9Hz, 1H), 1.94-1.84 (m, 2H), 1.66-1.45 (m, 5H), 0.97- 0.84 (m, 3H) 43

446.1/ 1.19 min (A)/ 0.21 μM δ 8.13 (br dd, J = 8.4, 4.4 Hz, 1H), 8.04(s, 1H), 7.73 (br s, 2H), 6.80 (d, J = 1.8 Hz, 1H), 4.64 (br d, J = 6.4Hz, 2H), 4.51 (br d, J = 14.0 Hz, 1H), 4.12 (br d, J = 13.4 Hz, 1H),3.98 (br d, J = 13.4 Hz, 1H), 3.11 (br d, J = 13.7 Hz, 1H), 2.64 (br s,1H), 2.33-2.20 (m, 1H), 2.12 (br d, J = 12.2 Hz, 1H), 2.08-1.95 (m, 2H),1.87 (br d, J = 6.7 Hz, 1H), 1.80 (br d, J = 11.0 Hz, 1H), 1.63 (br d, J= 12.8 Hz, 1H), 1.57- 1.41 (m, 3H), 0.98-0.87 (m, 6H) 44

444.2/ 1.16 min (A)/ 0.22 μM δ 8.12 (d, J = 8.5 Hz, 1H), 8.03 (s, 1H),7.74 (br d, J = 6.7 Hz, 2H), 6.80 (d, J = 2.1 Hz, 1H), 6.50 (br s, 1H),4.72-4.56 (m, 2H), 4.48 (br d, J = 12.5 Hz, 1H), 3.89 (br d, J = 12.8Hz, 1H), 3.78 (br d, J = 12.5 Hz, 1H), 3.18 (br s, 1H), 3.14-2.96 (m,1H), 2.88 (br t, J = 12.2 Hz, 1H), 2.63 (br s, 1H), 2.33-2.16 (m, 2H),2.16-2.05 (m, 2H), 1.99-1.90 (m, 1H), 1.92-1.89 (m, 1H), 1.89-1.73 (m,2H), 1.58 (br d, J = 11.3 Hz, 1H), 1.54-1.43 (m, 3H) 45

455.1/ 1.10 min (A)/ 0.11 μM δ 8.30 (br d, J = 8.5 Hz, 1H), 8.24 (br s,1H), 8.00 (br d, J = 8.5 Hz, 1H), 7.95 (s, 1H), 7.86 (br s, 1H), 6.85(s, 1H), 4.71 (br s, 2H), 4.46 (br s, 1H), 4.35 (br d, J = 13.4 Hz, 1H),3.18 (s, 1H), 2.89 (s, 1H), 2.73 (s, 1H), 2.19 (br d, J = 11.3 Hz, 1H),1.91 (m, 3H), 1.63 (br s, 3H), 1.49 (br t, J = 7.2 Hz, 4H) 46

466.1/ 1.18 min (A)/ 0.18 μM δ 8.30 (br s, 1H), 8.21 (br s, 1H), 7.95(s, 1H), 7.87 (br s, 1H), 7.47 (br s, 5H), 6.84 (br s, 1H), 4.76 (m,2H), 4.52 (br s, 1H), 3.83-3.59 (m, 1H), 3.31- 3.09 (m, 1H), 2.90 (s,1H), 2.74 (s, 1H), 2.19 (br d, J = 11.9 Hz, 1H), 1.99 (br s, 1H),1.92-1.63 (m, 2H), 1.55 (br s, 1.5H), 1.24 (br s, 1.5H) 47

448.1/ 0.97 min (A)/ 0.64 μM δ 8.13 (br dd, J = 8.4, 3.2 Hz, 1H), 8.04(s, 1H), 7.74 (br s, 2H), 6.80 (d, J = 2.1 Hz, 1H), 6.57 (br s, 1H),4.76-4.55 (m, 2H), 4.44 (br d, J = 12.5 Hz, 1H), 4.11 (br d, J = 12.2Hz, 1H), 3.99 (br d, J = 12.2 Hz, 1H), 3.63- 3.54 (m, 1H), 3.25 (d, J =10.4 Hz, 1H), 3.11 (br d, J = 10.7 Hz, 1H), 2.78-2.57 (m, 2H), 2.55 (s,3H), 2.17-2.04 (m, 1H), 1.98 (br d, J = 14.6 Hz, 1H), 1.94- 1.83 (m,1H), 1.77 (br d, J = 12.5 Hz, 1H), 1.65 (br d, J = 13.7 Hz, 1H),1.58-1.41 (m, 3H) 48

466.0/ 1.17 min (A)/ 0.08 μM δ 8.37-8.21 (m, 2H), 8.02 (br d, J = 8.5Hz, 1H), 7.87 (s, 1H), 6.89-6.83 (m, 1H), 4.70 (br s, 2H), 4.46 (br d, J= 11.6 Hz, 1H), 4.36 (br d, J = 13.1 Hz, 1H), 4.26- 4.00 (m, 1H),3.35-3.14 (m, 2H), 2.79 (br s, 1H), 2.18 (br s, 1H), 2.09-1.93 (m, 1H),1.88- 1.73 (m, 2H), 1.62 (br s, 1H), 1.50 (br t, J = 7.0 Hz, 3H) 49

428.1/ 1.02 min (A)/ 0.62 μM δ 8.12 (br d, J = 8.5 Hz, 1H), 8.03 (s,1H), 7.73 (br s, 2H), 6.79 (s, 1H), 6.47 (br s, 1H), 4.79 (s, 1H),4.71-4.54 (m, 2H), 4.47 (br d, J = 13.1 Hz, 1H), 3.90 (br s, 1H), 3.74(s, 1H), 2.39 (br d, J = 19.2 Hz, 1H), 2.27- 2.14 (m, 1H), 2.12 (br s,1H), 2.06 (br d, J = 8.2 Hz, 1H), 2.00- 1.84 (m, 1H), 1.84-1.72 (m, 1H),1.65 (br s, 1H), 1.58-1.39 (m, 3H), 1.31 (br d, J = 14.3 Hz, 1H) 50

480.1/ 1.27 min (A)/ 0.22 μM δ 8.90 (br s, 1H), 8.35-8.20 (m, 2H), 8.01(br d, J = 7.9 Hz, 1H), 7.88 (br s, 1H), 7.36-7.21 (m, 5H), 6.85 (s,1H), 4.75- 4.53 (m, 2H), 4.43 (br d, J = 6.7 Hz, 1H), 4.20 (br d, J =15.0 Hz, 1H), 4.08 (br d, J = 14.3 Hz, 1H), 3.91-3.74 (m, 2H), 3.24-3.05(m, 1H), 2.68 (br s, 1H), 2.12 (br s, 1H), 1.98 (br d, J = 11.6 Hz, 1H),1.92-1.74 (m, 1H), 1.64-1.45 (m, 3H), 1.39 (br t, J = 7.0 Hz, 1H) 51

418.2/ 1.14 min (A)/ 0.93 μM δ 8.23 (br d, J = 9.1 Hz, 1H), 8.04 (s,1H), 7.74 (br s, 1H), 6.80 (d, J = 2.0 Hz, 1H), 4.70- 4.52 (m, 1H),3.95-3.77 (m, 1H), 3.25-3.06 (m, 1H), 2.89 (s, 1H), 2.73 (s, 1H), 2.55(s, 2H), 2.50-2.27 (m, 1H), 2.08 (s, 3H), 1.91 (s, 3H), 1.84 (br d, J =13.1 Hz, 1H), 1.79-1.65 (m, 3H), 1.57 (br s, 1H) 52

446.2/ 1.40 min (A)/ 0.38 μM δ 8.23 (br d, J = 8.9 Hz, 1H), 8.03 (s,1H), 7.86-7.70 (m, 2H), 6.80 (d, J = 2.1 Hz, 1H), 6.51 (br s, 1H), 4.62(br d, J = 11.9 Hz, 1H), 4.07 (br d, J = 14.0 Hz, 1H), 3.74 (br d, J =16.8 Hz, 2H), 3.22-3.07 (m, 1H), 2.99-2.83 (m, 1H), 2.44- 2.23 (m, 2H),2.10 (br s, 1H), 1.98-1.81 (m, 3H), 1.81-1.68 (m, 5H), 1.63 (br s, 1H),1.53 (dq, J = 14.8, 7.3 Hz, 2H), 0.93- 0.83 (m, 3H) 53

460.3/ 1.36 min (A)/ 0.23 μM δ 8.23 (br d, J = 8.5 Hz, 1H), 8.04 (s,1H), 7.74 (br s, 2H), 6.93-6.78 (m, 1H), 6.49 (br s, 1H), 4.63 (br d, J= 11.6 Hz, 1H), 4.10 (br d, J = 14.3 Hz, 1H), 3.97 (br d, J = 12.5 Hz,1H), 3.66 (br s, 1H), 3.49 (br s, 1H), 3.19- 3.08 (m, 1H), 2.65 (br s,1H), 2.32-2.18 (m, 2H), 2.11 (br s, 1H), 2.07-1.94 (m, 2H), 1.91 (s,3H), 1.86 (br s, 1H), 1.82- 1.68 (m, 3H), 1.61 (br s, 2H), 1.22 (br s,1H), 0.95-0.84 (m, 3H) 54

458.1/ 1.28 min (A)/ 0.24 μM δ 8.23 (br d, J = 8.5 Hz, 1H), 8.05 (s,1H), 7.74 (br s, 2H), 6.80 (d, J = 1.8 Hz, 1H), 6.59 (br s, 1H), 4.60(br d, J = 11.3 Hz, 1H), 4.46 (br s, 1H), 4.01-3.83 (m, 1H), 3.77 (br d,J = 13.4 Hz, 1H), 3.55 (br s, 1H), 3.39 (br d, J = 8.5 Hz, 1H), 3.24 (brs, 1H), 3.17 (s, 1H), 3.13-2.98 (m, 1H), 2.64 (br d, J = 12.2 Hz, 1H),2.32-2.14 (m, 2H), 2.12 (br s, 2H), 2.06 (br s, 1H), 1.81-1.68 (m, 6H),1.57 (br s, 2H) 55

469.1/ 1.20 min (A)/ 0.23 μM δ 8.23 (br d, J = 8.5 Hz, 1H), 8.05 (s,1H), 7.85-7.66 (m, 2H), 6.83-6.77 (m, 1H), 6.54 (br s, 1H), 4.41 (br s,1H), 4.32 (br s, 1H), 3.63 (br s, 1H), 2.89 (s, 1H), 2.73 (s, 1H), 2.55(s, 1H), 2.15 (br s, 1H), 1.96 (br s, 1H), 1.91 (s, 2H), 1.74 (br s,6H), 1.61 (br s, 4H) 56

480.1/ 1.38 min (A)/ 0.30 μM δ 8.25 (br s, 1H), 8.12 (br s, 1H), 8.00(br s, 1H), 7.73 (br s, 2H), 7.48 (br s, 2H), 7.41 (br s, 3H), 6.80 (brd, J = 11.2 Hz, 1H), 3.39 (br s, 2H), 3.29-3.10 (m, 2H), 2.14 (br s,1H), 2.03 (br s, 1H), 1.90 (s, 3H), 1.77 (br s, 3H), 1.64 (br s, 2H),1.32 (br s, 2H) 57

494.2/ 1.50 min (A)/ 0.40 μM δ 8.21 (br dd, J = 19.7, 8.7 Hz, 1H), 8.05(br s, 1H), 7.75 (br s, 2H), 7.38-7.19 (m, 4H), 6.79 (s, 1H), 6.68-6.44(m, 1H), 4.63 (br d, J = 14.0 Hz, 1H), 4.53 (br s, 1H), 4.11 (br s, 1H),4.05 (br d, J = 12.2 Hz, 1H), 3.90- 3.69 (m, 1H), 3.58 (br d, J = 14.3Hz, 1H), 3.23-3.04 (m, 1H), 2.69 (br s, 1H), 2.08 (br s, 1H), 1.80 (brs, 1H), 1.74 (br d, J = 7.0 Hz, 3H), 1.63 (br d, J = 6.7 Hz, 3H), 1.57(br s, 2H) 58

494.2/ 1.29 min (A)/ 0.33 μM δ 8.23 (br d, J = 8.5 Hz, 1H), 8.05 (s,1H), 7.94 (s, 1H), 7.85- 7.70 (m, 2H), 6.85-6.74 (m, 1H), 4.58 (br d, J= 11.9 Hz, 1H), 4.45 (br s, 1H), 3.96 (br d, J = 12.2 Hz, 1H), 3.82 (brd, J = 13.1 Hz, 1H), 3.62 (br d, J = 14.6 Hz, 2H), 3.30 (br d, J = 8.9Hz, 1H), 3.23 (br s, 1H), 3.00 (br s, 1H), 2.83 (br s, 1H), 2.81-2.72(m, 1H), 2.10 (br s, 2H), 1.74 (br d, J = 6.4 Hz, 6H), 1.61 (br s, 2H)59

462.1/ 1.07 min (A)/ 0.97 μM δ 8.23 (br d, J = 8.6 Hz, 1H), 8.06 (s,1H), 7.76 (br s, 2H), 6.83-6.79 (m, 1H), 4.63 (br d, J = 14.4 Hz, 1H),4.10 (br d, J = 14.5 Hz, 1H), 3.99 (br d, J = 11.9 Hz, 1H), 3.69-3.49(m, 6H), 3.27-3.21 (m, 2H), 3.20- 3.01 (m, 1H), 2.65 (br d, J = 5.8 Hz,2H), 2.11 (br s, 1H), 1.86 (br d, J = 13.1 Hz, 1H), 1.75 (br d, J = 6.9Hz, 5H), 1.66 (br s, 1H), 1.56 (br s, 1H) 60

404.3/ 1.16 min (B)/ 4.6 μM δ 8.31-8.08 (m, 2H), 7.99- 7.89 (m, 1H),7.87-7.75 (m, 1H), 6.89-6.77 (m, 1H), 3.05- 2.85 (m, 1H), 2.33-2.18 (m,1H), 2.05-1.86 (m, 2H), 1.63- 1.42 (m, 3H), 1.23-1.11 (m, 2H), 1.11-0.87(m, 7H) 61

418.1/ 1.13 min (B)/ 2.3 μM δ 8.28-8.16 (m, 2H), 8.05- 7.96 (m, 1H),7.90-7.81 (m, 1H), 6.89-6.81 (m, 1H), 4.76- 4.64 (m, 1H), 4.40-4.26 (m,1H), 3.70-3.61 (m, 1H), 3.11- 3.03 (m, 1H), 3.02-2.86 (m, 1H), 2.33-2.21(m, 1H), 2.00- 1.81 (m, 2H), 1.63-1.48 (m, 1H), 1.29-1.19 (m, 9H) 62

404.3/ 1.17 min (B)/ 1.2 μM δ 8.30-8.11 (m, 2H), 8.04- 7.91 (m, 1H),7.88-7.76 (m, 1H), 6.90-6.78 (m, 1H), 4.43- 4.10 (m, 1H), 2.99-2.85 (m,1H), 2.56-2.54 (m, 1H), 2.42- 2.19 (m, 3H), 1.99-1.78 (m, 2H), 1.65-1.43(m, 4H), 1.22- 1.10 (m, 1H), 0.99-0.81 (m, 3H) 63

437.8/ 1.24 min (B)/ 6.6 μM δ 8.35-8.17 (m, 2H), 8.10- 7.97 (m, 1H),7.93-7.80 (m, 1H), 6.90-6.77 (m, 1H), 4.86- 4.71 (m, 1H), 4.22-4.07 (m,1H), 3.54-3.40 (m, 2H), 2.99- 2.81 (m, 1H), 2.43-2.18 (m, 1H), 2.08-1.68(m, 4H), 1.60- 1.41 (m, 1H), 1.21-1.11 (m, 2H) 64

406.2/ 0.85 min (B)/ 9.9 μM δ 8.26-8.13 (m, 2H), 8.04- 7.95 (m, 1H),7.89-7.78 (m, 1H), 6.91-6.78 (m, 1H), 4.79- 4.65 (m, 1H), 4.32-4.01 (m,3H), 3.67-3.51 (m, 1H), 3.34- 3.25 (m, 1H), 3.11-3.00 (m, 1H), 2.97-2.85(m, 1H), 2.57- 2.54 (m, 2H), 2.33-2.19 (m, 1H), 2.03-1.76 (m, 2H), 1.68-1.43 (m, 1H), 1.21-1.10 (m, 1H) 65

400.7/ 1.15 min (B)/ 0.35 μM δ 8.29-8.13 (m, 2H), 8.06- 7.96 (m, 1H),7.90-7.76 (m, 1H), 6.93-6.80 (m, 1H), 6.44- 6.32 (m, 1H), 5.34-5.16 (m,1H), 4.83-4.66 (m, 1H), 4.43- 4.06 (m, 1H), 3.24-3.00 (m, 1H), 2.98-2.75(m, 1H), 2.59- 2.54 (m, 2H), 2.35-2.21 (m, 1H), 2.02-1.74 (m, 2H), 1.68-1.43 (m, 1H), 1.28-1.20 (m, 1H) 66

418.1/ 1.28 min (B)/ 3.5 μM δ 8.11-8.03 (m, 1H), 8.02- 7.94 (m, 1H),7.76-7.66 (m, 2H), 6.83-6.74 (m, 1H), 3.15- 3.04 (m, 1H), 3.03-2.82 (m,1H), 2.34-2.18 (m, 3H), 2.05- 1.94 (m, 1H), 1.94-1.86 (m, 4H), 1.84-1.75(m, 1H), 1.59- 1.38 (m, 1H), 0.98-0.78 (m, 6H) 67

438.2/ 1.11 min (B)/ 1.8 μM δ 8.32-8.08 (m, 2H), 8.03- 7.91 (m, 1H),7.89-7.74 (m, 1H), 7.57-7.31 (m, 5H), 6.91- 6.77 (m, 1H), 3.65-3.44 (m,4H), 2.37-2.24 (m, 1H), 2.05- 1.88 (m, 1H), 1.90-1.76 (m, 1H), 1.76-1.58(m, 1H), 1.58- 1.42 (m, 1H) 68

470.1/ 1.29 min (B)/ 2.3 μM δ 8.34-8.18 (m, 2H), 8.07- 7.97 (m, 1H),7.93-7.81 (m, 1H), 6.91-6.73 (m, 1H), 4.86- 4.56 (m, 1H), 4.46-4.21 (m,1H), 3.99-3.91 (m, 1H), 3.00- 2.86 (m, 1H), 2.41-2.23 (m, 1H), 2.07-1.87(m, 2H), 1.76- 1.53 (m, 1H), 1.39-1.31 (m, 2H), 1.30-1.20 (m, 2H), 1.20-1.14 (m, 1H) 69

441.2/ 1.21 min (B)/ 2.0 μM δ 8.11-8.03 (m, 1H), 8.01- 7.96 (m, 1H),7.79-7.65 (m, 2H), 6.82-6.74 (m, 1H), 3.74- 3.58 (m, 4H), 2.89-2.68 (m,2H), 2.65-2.54 (m, 4H), 2.38- 2.24 (m, 1H), 2.21-2.09 (m, 1H), 2.00-1.92(m, 2H), 1.88- 1.81 (m, 1H) 70

455.2/ 1.35 min (B)/ 3.9 μM TLR7 EC₅₀ 57 μM δ 8.27-8.13 (m, 2H), 8.07-7.95 (m, 1H), 7.90-7.79 (m, 1H), 6.89-6.82 (m, 1H), 4.83- 4.63 (m, 1H),4.57-4.41 (m, 1H), 4.26-4.09 (m, 1H), 3.74- 3.51 (m, 2H), 2.43-2.14 (m,5H), 2.05-1.83 (m, 2H), 1.82- 1.62 (m, 5H) 71

362.3/ 0.79 min (A)/ 4.9 μM δ 8.22 (br d, J = 8.2 Hz, 2H), 8.01 (br d, J= 7.9 Hz, 1H), 7.86 (br s, 1H), 6.83 (s, 1H), 3.95- 3.78 (m, 1H), 3.71(br d, J = 8.5 Hz, 1H), 3.64-3.58 (m, 1H), 2.89 (s, 1H), 2.73 (s, 1H),2.46- 2.34 (m, 1H), 2.28 (br d, J = 8.9 Hz, 1H), 2.00 (d, J = 6.7 Hz,3H) 72

390.1/ 0.84 min (A)/ 3.1 μM δ 8.21 (br d, J = 7.3 Hz, 2H), 8.01 (br d, J= 8.2 Hz, 1H), 7.85 (br s, 1H), 6.83 (s, 1H), 3.96- 3.85 (m, 1H), 3.79(br d, J = 8.2 Hz, 1H), 3.75-3.60 (m, 1H), 2.89 (s, 1H), 2.75-2.68 (m,2H), 2.45-2.33 (m, 1H), 2.27 (br d, J = 8.2 Hz, 1H), 1.07-0.99 (m, 6H)73

434.4/ 0.71 min (C)/ 9.0 μM δ 8.02 (br d, J = 8.2 Hz, 1H), 7.96 (s, 1H),7.67 (br d, J = 8.2 Hz, 1H), 6.75 (s, 1H), 6.52 (br s, 1H), 4.00-3.77(m, 2H), 3.70 (br s, 2H), 3.21-3.02 (m, 2H), 2.87 (s, 1H), 1.87 (s, 3H),1.20 (s, 9H) 74

430.0/ 0.98 min (A)/ 6.8 μM δ 8.03 (br s, 1H), 7.99 (s, 1H), 7.77-7.66(m, 2H), 6.77 (s, 1H), 6.59 (br s, 1H), 4.05 (br s, 1H), 3.98-3.88 (m,1H), 3.85 (br d, J = 5.8 Hz, 1H), 3.75 (br d, J = 6.4 Hz, 2H), 3.68-3.51(m, 2H), 3.21-3.04 (m, 1H), 2.37 (br s, 1H) 75

413.4/ 0.92 min (A)/ 5.0 μM δ 8.21 (br s, 2H), 8.00 (br d, J = 7.9 Hz,1H), 7.85 (br s, 1H), 6.84 (s, 1H), 4.05 (br s, 1H), 3.95 (br s, 1H),3.90 (br s, 1H), 3.84 (br d, J = 9.8 Hz, 1H), 2.43 (br d, J = 7.6 Hz,1H), 2.35 (br d, J = 8.2 Hz, 1H), 1.66-1.56 (m, 3H), 1.52 (br s, 1H),1.47 (br s, 1H) 76

424.0/ 0.94 min (A)/ 5.5 μM δ 8.22 (br s, 2H), 8.02 (br s, 1H), 7.83 (s,1H), 7.56 (d, J = 6.6 Hz, 2H), 7.52-7.44 (m, 3H), 6.82 (br s, 1H), 4.11(br s, 1H), 3.97 (br s, 1H), 3.87 (br s, 1H), 3.81 (br s, 1H), 3.68 (brs, 1H), 2.48-2.28 (m, 2H) 77

423.9/ 1.03 min (A)/ 4.1 μM δ 8.17-7.86 (m, 3H), 7.76- 7.63 (m, 2H),7.62-7.53 (m, 2H), 7.52-7.43 (m, 3H), 6.80- 6.68 (m, 1H), 4.17-4.01 (m,1H), 4.05-3.73 (m, 3H), 3.71- 3.60 (m, 1H), 2.44-2.28 (m, 1H) 78

404.1/ 1.29 min (B)/ 5.8 μM δ 8.06-7.99 (m, 1H), 7.98- 7.95 (m, 1H),7.74-7.65 (m, 2H), 6.80-6.74 (m, 1H), 4.47- 4.37 (m, 1H), 3.62-3.46 (m,3H), 3.44-3.30 (m, 2H), 2.33- 2.17 (m, 2H), 1.87-1.77 (m, 4H), 1.60-1.49(m, 2H), 0.93- 0.85 (m, 3H) 79

404.3/ 1.08 min (B)/ 1.6 μM δ 9.09-8.61 (m, 1H), 8.27- 8.09 (m, 2H),8.05-7.93 (m, 1H), 7.89-7.77 (m, 1H), 6.87- 6.79 (m, 1H), 4.48-4.40 (m,1H), 3.63-3.36 (m, 1H), 2.98- 2.82 (m, 1H), 2.75-2.59 (m, 1H), 2.54 (s,1H), 2.08-1.79 (m, 4H), 1.61-1.41 (m, 1H), 1.06-0.91 (m, 6H) 80

406.2/ 0.85 min (B)/ 6.6 μM δ 9.35-8.63 (m, 1H), 8.33- 8.10 (m, 2H),8.07-7.91 (m, 1H), 7.90-7.76 (m, 1H), 7.45- 6.94 (m, 1H), 6.94-6.77 (m,1H), 4.54-4.44 (m, 1H), 4.24- 3.81 (m, 2H), 3.63-3.42 (m, 4H), 3.34-3.23(m, 1H), 3.02- 2.85 (m, 1H), 2.01-1.72 (m, 4H) 81

470.0/ 1.25 min (B)/ 4.0 μM δ 9.03-8.72 (m, 1H), 8.27- 8.12 (m, 2H),8.06-7.93 (m, 1H), 7.91-7.81 (m, 1H), 6.88- 6.79 (m, 1H), 4.52-4.40 (m,1H), 3.77-3.67 (m, 1H), 3.60- 3.27 (m, 1H), 3.03-2.85 (m, 1H), 1.97 (brd, J = 4.9 Hz, 2H), 1.92-1.76 (m, 2H), 1.31-1.20 (m, 4H), 1.20-1.13 (m,2H) 82

441.3/ 1.12 min (B)/ 2.6 μM δ 8.97-8.60 (m, 1H), 8.26- 8.06 (m, 2H),8.07-7.92 (m, 1H), 7.91-7.76 (m, 1H), 6.88- 6.79 (m, 1H), 4.55-4.42 (m,1H), 3.54-3.27 (m, 2H), 3.21- 3.11 (m, 3H), 3.12-3.01 (m, 1H), 2.97-2.85(m, 1H), 2.82- 2.70 (m, 1H), 2.68-2.56 (m, 1H), 2.19-2.06 (m, 1H), 2.04-1.79 (m, 5H) 83

455.3/ 1.32 min (B)/ 6.6 μM δ 8.09-7.99 (m, 2H), 7.77- 7.70 (m, 2H),6.80-6.75 (m, 1H), 4.52-4.43 (m, 1H), 3.90- 3.86 (m, 1H), 3.57 (br d, J= 9.2 Hz, 2H), 3.18-3.14 (m, 1H), 2.41-2.12 (m, 3H), 2.02-1.92 (m, 1H),1.92-1.82 (m, 2H), 1.75-1.61 (m, 2H), 1.57-1.43 (m, 1H), 1.27-1.17 (m,2H), 0.97-0.85 (m, 1H) 84

438.2/ 1.09 min (B)/ 2.5 μM δ 9.24-8.61 (m, 3H), 8.28- 8.15 (m, 2H),8.04-7.97 (m, 1H), 7.88-7.81 (m, 1H), 7.56- 7.48 (m, 2H), 7.48-7.39 (m,3H), 6.87-6.80 (m, 1H), 4.65 (br d, J = 3.1 Hz, 1H), 3.64-2.87 (m, 4H),2.10-1.96 (m, 1H), 1.96-1.83 (m, 2H), 1.83-1.68 (m, 1H) 85

439.1/ 1.08 min (B)/ 5.9 μM δ 8.61-8.53 (m, 1H), 8.06- 7.98 (m, 1H),7.98-7.82 (m, 3H), 7.72-7.62 (m, 4H), 7.49- 7.43 (m, 1H), 6.77-6.70 (m,1H), 4.68-4.58 (m, 1H), 3.73- 3.27 (m, 2H), 3.00-2.88 (m, 1H), 2.86-2.80(m, 1H), 2.72- 2.64 (m, 1H), 1.96-1.88 (m, 3H), 1.81-1.70 (m, 1H) 86

404.1/ 1.36 min (B)/ 6.4 μM δ 8.03 (br d, J = 5.8 Hz, 1H), 7.98 (s, 1H),7.75-7.66 (m, 2H), 6.80-6.75 (m, 1H), 4.45- 4.37 (m, 1H), 3.61-3.45 (m,2H), 3.35-3.25 (m, 1H), 2.91- 2.81 (m, 1H), 2.73-2.62 (m, 1H), 1.99-1.79(m, 4H), 1.06- 0.95 (m, 6H) 87

418.2/ 1.11 min (B)/ 4.3 μM TLR8 EC₅₀ 77 μM δ 9.06-8.65 (m, 2H), 8.27-8.11 (m, 2H), 8.05-7.93 (m, 1H), 7.92-7.80 (m, 1H), 6.87- 6.77 (m, 1H),4.52-4.40 (m, 1H), 3.72-3.25 (m, 2H), 3.23- 2.76 (m, 2H), 2.23-1.78 (m,7H), 0.94-0.84 (m, 6H) 88

418.4/ 1.55 min (B)/ 1.9 μM δ 8.28-8.13 (m, 2H), 8.05- 7.91 (m, 1H),7.91-7.80 (m, 1H), 6.86-6.80 (m, 1H), 4.56- 4.44 (m, 1H), 3.73-3.52 (m,1H), 3.37-3.29 (m, 2H), 2.95- 2.81 (m, 1H), 2.07-1.78 (m, 4H), 1.21-1.14(m, 9H) 89

416.2/ 1.22 min (B)/ 2.8 μM δ 9.08-8.67 (m, 2H), 8.27- 8.13 (m, 2H),8.06-7.95 (m, 1H), 7.90-7.81 (m, 1H), 6.89- 6.79 (m, 1H), 4.48-4.37 (m,1H), 3.58-3.46 (m, 3H), 3.42- 3.21 (m, 1H), 2.96-2.81 (m, 1H), 2.21-1.99(m, 4H), 1.98- 1.68 (m, 6H) 90

420.1/ 1.17 min (B)/ 6.0 μM δ 8.87-8.49 (m, 2H), 8.24- 8.06 (m, 2H),8.02-7.89 (m, 1H), 7.87-7.76 (m, 1H), 6.87- 6.76 (m, 1H), 4.50-4.38 (m,1H), 3.59-3.31 (m, 4H), 3.23- 3.17 (m, 3H), 2.95-2.81 (m, 1H), 2.74-2.65(m, 1H), 2.02- 1.69 (m, 6H) 91

438.1/ 1.14 min (B)/ 4.1 μM δ 9.26-8.60 (m, 2H), 8.22- 8.07 (m, 2H),8.00-7.89 (m, 1H), 7.85-7.75 (m, 1H), 6.84- 6.76 (m, 1H), 4.49-4.37 (m,1H), 3.50 (br s, 2H), 2.95 (s, 2H), 2.10-1.72 (m, 7H) 92

427.1/ 1.14 min (B)/ 9.4 μM δ 8.98-8.64 (m, 2H), 8.24- 8.07 (m, 2H),7.99-7.90 (m, 1H), 7.86-7.73 (m, 1H), 6.84- 6.75 (m, 1H), 4.44-4.35 (m,1H), 3.80-3.62 (m, 1H), 3.48- 3.22 (m, 2H), 3.07-2.95 (m, 1H), 2.00-1.79(m, 3H), 1.62- 1.53 (m, 3H), 1.42 (br d, J = 5.2 Hz, 2H) 93

470.2/ 1.25 min (B)/ 3.2 μM δ 9.11-8.55 (m, 2H), 8.24- 8.05 (m, 2H),8.00-7.89 (m, 1H), 7.84-7.75 (m, 1H), 6.84- 6.75 (m, 1H), 4.46-4.36 (m,1H), 3.74-3.63 (m, 1H), 3.57- 3.49 (m, 2H), 3.01-2.86 (m, 1H), 2.01-1.72(m, 3H), 1.27- 1.14 (m, 3H), 0.89 (s, 2H) 94

441.2/ 1.39 min (B)/ 6.2 μM δ 9.01-8.49 (m, 2H), 8.22- 8.10 (m, 2H),8.03-7.95 (m, 1H), 7.86-7.79 (m, 1H), 6.88- 6.80 (m, 1H), 4.53-4.43 (m,1H), 3.70-3.60 (m, 2H), 3.45- 3.35 (m, 1H), 3.13-3.03 (m, 1H), 2.81-2.68(m, 2H), 2.64- 2.55 (m, 2H), 2.19-2.06 (m, 1H), 2.04-1.94 (m, 1H), 1.94-1.79 (m, 4H) 95

455.2/ 1.52 min (B)/ 6.5 μM δ 8.20-8.15 (m, 1H), 8.12 (s, 1H), 7.88-7.81(m, 2H), 6.92- 6.88 (m, 1H), 4.66-4.57 (m, 1H), 3.92-3.80 (m, 1H), 3.47-3.34 (m, 1H), 3.25-3.09 (m, 1H), 2.56-2.27 (m, 4H), 2.15- 1.95 (m, 5H),1.93-1.76 (m, 4H) 96

438.2/ 1.11 min (B)/ 11 μM δ 8.08-8.00 (m, 1H), 8.00- 7.95 (m, 1H),7.72-7.64 (m, 2H), 7.53-7.46 (m, 2H), 7.45- 7.33 (m, 3H), 6.78-6.71 (m,1H), 4.62-4.51 (m, 1H), 3.56- 3.39 (m, 2H), 3.30-2.96 (m, 2H), 2.05-1.90(m, 1H), 1.83- 1.62 (m, 3H)

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A compound of Formula (I):

or a stereoisomer, a tautomer or a pharmaceutically acceptable saltthereof, wherein: R¹ is independently selected from: H or C₁₋₆ alkylsubstituted with 0 to 3 halogen; R², R³ and R⁵ are, at each occurrence,independently selected from: H, halogen, cyano, C₁₋₄ alkyl, C₁₋₄haloalkyl, C₁₋₄ alkoxy, and C₁₋₄ haloalkoxy; R⁴ is independently5-membered heteroaryl including from 1 to 4 ring atoms are eachindependently selected from N, N(R^(a)), O, and S, wherein theheteroaryl is substituted with from 0 to 3 R^(b); R⁶ is independentlyselected from:

R⁷ is independently selected from: H, C₁₋₄ alkyl and —C(═O)R⁸; R⁸ isindependently selected from: C₂₋₄ alkynyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl,C₁₋₄ alkyl substituted with 0 to 2 R^(c), —(CH₂)₀₋₂—C₃₋₆ cycloalkylsubstituted with 0 to 3 R^(d), —(CH₂)₀₋₂-phenyl substituted with 0 to 3R^(d), and —(CH₂)₀₋₂-(5- to 6-membered heteroaryl including from 1 to 4ring atoms are each independently selected from N, N(R^(a)), O, and S,wherein the heteroaryl is substituted with from 0 to 3 R^(d)); R^(a) is,at each occurrence, independently selected from: H and C₁₋₄ alkyl; andR^(b), R^(c) and R^(d) are, at each occurrence, independently selectedfrom: halogen, OH, cyano, C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₁₋₄ alkoxy, andC₁₋₄ haloalkoxy.
 2. The compound of claim 1, wherein: R¹ isindependently selected from: H or C₁₋₆ alkyl; R², R³ and R⁵ are, at eachoccurrence, independently selected from: H, halogen and C₁₋₄ alkyl; R⁴is independently pyrazolyl, thienyl or isothiazolyl; R⁶ is independentlyselected from:

and R⁷ is independently H or —C(═O)R⁸.
 3. The compound of claim 2,wherein the compound is of Formula (II):

or a stereoisomer, a tautomer or a pharmaceutically acceptable saltthereof; wherein: R¹ is independently H or C₁₋₄ alkyl; R⁶ isindependently selected from:

R⁸ is independently selected from: C₂₋₄ alkynyl, C₁₋₄ alkoxy, C₁₋₄haloalkyl, phenyl, pyridyl, benzyl, C₁₋₄ alkyl substituted with 0 to 1R^(c), and C₃₋₆ cycloalkyl substituted with 0 to 2 R^(d); R^(c) isindependently C₁₋₄ alkoxy or cyano; and R^(d) is independently selectedfrom: F, cyano and C₁₋₄ haloalkyl.
 4. The compound of claim 3, wherein:R⁶ is independently


5. The compound of claim 3, wherein: R⁶ is independently selected from:


6. The compound of claim 4, wherein: R⁶ is independently


7. A compound according to claim 1, wherein the compound is selectedfrom Examples 1 to 96 or a pharmaceutically acceptable salt thereof. 8.A pharmaceutical composition comprising a compound or a pharmaceuticallyacceptable salt thereof as claimed in claim 1 and one or morepharmaceutically acceptable excipients.
 9. (canceled)
 10. A method oftreating cancer, comprising administering to a subject in need of suchtreatment an effective amount of a compound or a pharmaceuticallyacceptable salt thereof as claimed in claim
 1. 11. The method of claim10, wherein the cancer is selected from acute myeloid leukemia,adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer,appendix cancer, teratoid/rhabdoid tumor, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancer, brain cancer, breast cancer,bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer,chordoma, chronic lymphocytic leukemia, chronic myeloproliferativeneoplasm, colon cancer, colorectal cancer, craniopharyngioma,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, eye cancer, fallopian tube cancer,gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor, germ cell tumor, hairy cell leukemia, head and neckcancer, heart cancer, liver cancer, hypopharngeal cancer, pancreaticcancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia,lip and oral cavity cancer, lung cancer, melanoma, Merkel cellcarcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma,ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer,rectal cancer, salivary gland cancer, skin cancer, small intestinecancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, and vulvarcancer.
 12. The method of claim 10, wherein the cancer is selected frombreast cancer, colon cancer, rectal cancer, colorectal cancer,pancreatic cancer, and prostate cancer.
 13. The method of claim 10,wherein the cancer is selected from hormone receptor positive breastcancer, microsatellite stable colon or rectal cancer, pancreatic cancerand prostate cancer.
 14. The method of claim 9, wherein the compound isadministered in combination with one or more additional cancertherapies.
 15. The method of claim 14, wherein the one or moreadditional cancer therapies comprise surgery, radiotherapy,chemotherapy, toxin therapy, immunotherapy, cryotherapy or gene therapy,or a combination thereof.
 16. The method of claim 14, wherein theadditional cancer therapy comprises one or more agents selected fromnivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001,BGB-A317, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034,MGD013, IBI308, BMS-936559, atezolizumab, durvalumab, avelumab,STI-1014, CX-072, LY3300054, CK-301, urelumab, PF-05082566, MEDI6469,TRX518, varlilumab, CP-870893, BMS-986016, MGA271, lirilumab, IPH2201,emactuzumab, INCB024360, galunisertib, ulocuplumab, BKT140, Bavituximab,CC-90002, bevacizumab, MNRP1685A, ipilimumab, MK-1308, AGEN-1884, andtremelimumab.
 17. The method of claim 14, wherein the additional cancertherapy comprises one or more agents selected from nivolumab,ipilimumab, pembrolizumab, atezolizumab, durvalumab and avelumab.